Canonical NF-κB Pathway Research Articles

Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor-κB inflammatory signaling pathway in human skin cells.

Introduction Corynebacterium tuberculostearicum (C. t.) is a ubiquitous bacterium that colonizes human skin. In contrast to other members of the genus Corynebacterium, such as toxigenic Corynebacterium diphtheriae or the opportunistic pathogen Corynebacterium jeikeium, several studies suggest that C. t. may play a role in skin health and disease. However, the mechanisms underlying these effects remain poorly understood.MethodsTo investigate whether C. t. induces inflammatory pathways in primary human epidermal keratinocytes (HEKs) and human cutaneous squamous carcinoma cells (SCCs), cell culture, reverse transcription‐polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay, immunofluorescence microscopy, Western blot, chromatin immunoprecipitation‐PCR, small interfering RNA knockdown and luciferase reporter expression system were used.ResultsHerein, we demonstrate that C. t. upregulates the messenger RNA (mRNA) and protein levels of inflammatory mediators in two human skin cell lines, HEKs and SCCs. We further show activation of the canonical nuclear factor‐κB (NF‐κB) pathway in response to C. t. infection, including phosphorylation of the inhibitor of κB (IκB), the nuclear translocation of NF‐κB subunit (NF‐κB‐P65) and the recruitment of NF‐κB‐P65 and RNA polymerase to the NF‐κB response elements at the promoter region of the inflammatory genes. Lastly, the data confirm that C. t.‐induced tumor necrosis factor mRNA expression in HEKs is toll‐like receptor 2 (TLR2) dependent.ConclusionOur results offer a mechanistic model for C. t.‐induced inflammation in human keratinocytes via TLR2 and activation of IκB kinase and downstream signaling through the canonical NF‐κB pathway. Relevance to chronic inflammatory diseases of the skin and cutaneous oncology is discussed.

Molecular Mechanisms of Notch Signaling in Lymphoid Cell Lineages Development: NF-κB and Beyond.

Notch is a ligand-receptor interaction-triggered signaling cascade highly conserved, that influences multiple lineage decisions within the hematopoietic and the immune system. It is a recognized model of intercellular communication that plays an essential role in embryonic as well as in adult immune cell development and homeostasis. Four members belong to the family of Notch receptors (Notch1-4), and each of them plays nonredundant functions at several developmental stages. Canonical and noncanonical pathways of Notch signaling are multifaceted drivers of immune cells biology. In fact, increasing evidence highlighted Notch as an important modulator of immune responses, also in cancer microenvironment. In these contexts, multiple transduction signals, including canonical and alternative NF-κB pathways, play a relevant role. In this chapter, we will first describe the critical role of Notch and NF-κB signals in lymphoid lineages developing in thymus: natural killer T cells, thymocytes, and thymic T regulatory cells. We will address also the role played by ligand expressing cells. Given the importance of Notch/NF-κB cross talk, its role in T-cell leukemia development and progression will be discussed.

NF-κB Inhibition Suppresses Experimental Melanoma Lung Metastasis

Background:Although novel therapeutic regimens for melanoma continue to emerge, the best current clinical response rate is still less than 60%. Moreover, antimelanoma treatments contribute to toxicities in other vital organs. In this study, we elucidate the therapeutic advantages of siRNA targeting melanoma NF-κB canonical signaling pathway with a peptide-based gene delivery nanoplex system.Methods and Results:In vitro treatment of melanoma B16-F10 cells was used to demonstrate delivery and efficacy of anti-NF-kB siRNA to cell cytoplasm with a 55 mn peptide-based gene delivery system. NF-κB (p65) knockdown was validated both at mRNA and protein levels by using RT2-PCR, western blot, and immunofluorescence cellular staining. Canonical p65 mRNA was reduced by 82% and p65 protein was reduced by 48%, which differed significantly from levels in control groups. In vivo treatment of a melanoma lung metastasis mouse model with 3-serial i.v. injections of p5RHH-p65 siRNA nanoparticles retarded growth of lung metastasis within one week by 76% (p=0.003) as compared to saline control treatments.Conclusion:Inhibition of melanoma NF-κB (p65) with systemically-delivered siRNA effectively impedes the growth and progression of experimental melanoma lung metastasis.

GYS1 induces glycogen accumulation and promotes tumor progression via the NF-κB pathway in Clear Cell Renal Carcinoma.

Metabolism reprogramming is a hallmark of many cancer types. We focused on clear cell renal carcinoma (ccRCC) which is characterized by its clear and glycogen-enriched cytoplasm with unknown reasons. The aim of this study was to identify the clinical significance, biological function, and molecular regulation of glycogen synthase 1 (GYS1) in ccRCC glycogen accumulation and tumor progression.Methods: We determined the clinical relevance of GYS1 and glycogen in ccRCC by immunohistochemistry and periodic acid-schiff staining in fresh tissue and by tissue micro-array. Metabolic profiling with GYS1 depletion was performed by metabolomics analysis. In vitro and xenograft mouse models were used to evaluate the impact of GYS1 on cell proliferation. High-throughput RNA-Seq analyses and co-immunoprecipitation-linked mass spectrometry were used to investigate the downstream targets of GYS1. Flow cytometry and CCK8 assays were performed to determine the effect of GYS1 and sunitinib on cell viability.Results: We observed that GYS1 was significantly overexpressed and glycogen was accumulated in ccRCC tissues. These effects were correlated with unfavorable patient survival. Silencing of GYS1 induced metabolomic perturbation manifested by a carbohydrate metabolism shift. Overexpression of GYS1 promoted tumor growth whereas its silencing suppressed it by activating the canonical NF-κB pathway. The indirect interaction between GYS1 and NF-κB was intermediated by RPS27A, which facilitated the phosphorylation and nuclear import of p65. Moreover, silencing of GYS1 increased the synthetic lethality of ccRCC cells to sunitinib treatment by concomitantly suppressing p65.Conclusions: Our study findings reveal an oncogenic role for GYS1 in cell proliferation and glycogen metabolism in ccRCC. Re-sensitization of ccRCC cells to sunitinib suggests that GYS1 is a useful indicator of unfavorable prognosis as well as a therapeutic target for patients with ccRCC.

TB-02 NF-KB CANONICAL PATHWAY ACTIVATION DRIVES GLYCOLYSIS AND TUMOR PROGRESSION IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Recent genomic analyses have identified highly recurrent genetic alterations in PCNSL. However, due to the lack of clinically representative PCNSL preclinical models, the pathogenic mechanisms of these alterations remains largely unknown. Here, we established the largest panel of 12 clinically relevant PCNSL patient-derived orthotopic xenografts retained the histopathologic phenotype, lymphoma expression subtype, copy number alterations and 90% of the non-synonymous mutations of primary tumors, with 100% concordance of MYD88 and CD79B mutations, which are highly recurrent in PCNSL. Patient tumor regression with high-dose methotrexate correlated with in vitro sensitivity to methotrexate in corresponding PCNSL models. By knocking down canonical NF-kB pathway genes, we found that successful orthotopic xenograft formation was dependent on NF-kB canonical pathway activation induced by MYD88 mutation or overexpression of EBV-related LMP1. Metabolically, PCNSL xenografts phenocopied the high 18F-fluorodeoxyglucose uptake observed in patients and demonstrated glycolytic dependence, revealing new potential therapeutic strategies in PCNSL. Collectively, we found NF-kB canonical pathway activation as a crucial driver of PCNSL xenograft progression and found that NF-kB canonical pathway induced an addiction to glycolysis, revealing a novel potential therapeutic strategy. Our PCNSL xenograft panel represents a valuable and reproducible preclinical tool that has the potential to help decipher how genetic and/or epigenetic alterations contributes to lymphomagenesis and tumor maintenance and enhance the development of novel therapeutic strategies in PCNSL.

NF-κB-Mediated Neuroinflammation in Parkinson's Disease and Potential Therapeutic Effect of Polyphenols.

Different animal and human studies from last two decades in the case of Parkinson's disease (PD) have concentrated on oxidative stress due to increased inflammation and cytokine-dependent neurotoxicity leading to induction of dopaminergic (DA) degeneration pathway in the nigrostriatal region. Chronic inflammation, the principle hallmark of PD, forms the basis of neurodegeneration. Aging in association with activation of glia due to neuronal injury, perhaps because of immune alterations and genetic predispositions, leads to deregulation of inflammatory pathways premising the onset of PD. A family of inducible transcription factors, nuclear factor-κB (NF-κB), is found to show expression in various cells and tissues, such as microglia, neurons, and astrocytes which play an important role in activation and regulation of inflammatory intermediates during inflammation. Both canonical and non-canonical NF-κB pathways are involved in the regulation of the stimulated cells. During the prodromal/asymptomatic stage of age-associated neurodegenerative diseases (i.e., PD and AD), chronic neuroinflammation may act silently as the driver of neuronal dysfunction. Though research has provided an insight over age-related neurodegeneration in PD, elaborative role of NF-κB in neuroinflammation is yet to be completely understood and thus requires more investigation. Polyphenols, a group of naturally occurring compound in medicinal plants, have gained attention because of their anti-oxidative and anti-neuroinflammatory properties in neurodegenerative diseases. In this aspect, this review highlights the role of NF-κB and the possible therapeutic roles of polyphenols in NF-κB-mediated neuroinflammation in PD.

TNF-α Confers Resistance of Myeloma Cells to IMiDs through TRAF2 Degradation

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse during the course of treatment. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Therefore, CRBN is the primary binding target and master regulator of IMiDs sensitivity; however, the molecular mechanisms regulating resistance to IMiDs have not been fully defined. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. To delineate the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. We observed that knockout (KO) of twenty-eight genes and one miRNA were associated with resistance to IMiDs. Of note, all six sgRNAs targeting CRBN were identified as a positive regulator of IMiDs sensitivity, consistent with previous studies. Among these genes, we found that three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Therefore, we next individually cloned the sgRNAs of TRAF2 into the Cas9 lentiviral vector, and then re-introduced them into MM.1S cells. Importantly, TRAF2-KO MM.1S cells acquire significant resistance to Pom and Len treatment. To examine whether TRAF2 KO induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with downregulation of IRF4. Taken together, these data suggest that TRAF2 mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis. Since TRAF2 is a member of the TNF receptor associated factor (TRAF) protein family required for activation of several signaling pathways including NF-ĸB and JNK, we next examined the biologic impact of TRAF2 KO. Importantly, TRAF2 KO cells show significantly increased processing of precursor p100 to p52 (NF-ĸB2), resulting in hyperactivation of the non-canonical NF-κB pathway; conversely, p52 KO re-sensitizes the cells to IMiDs treatment. The activity of the canonical NF-κB pathway was not similarly altered in TRAF2 KO cells. These results suggest that TRAF2 predominantly activates non-canonical NF-κB pathway, associated with resistance to IMiDs in MM cells. We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either bone marrow stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 is downregulated in BMSCs and BM-CS, suggesting that IMiDs resistance in the BM microenvironment may be mediated by TRAF2 downregulation induced by soluble factors. Cytokine array assay confirmed detectable TNF-α in BM-CS. Indeed, MM cells treated with TNF-α showed decreased TRAF2 expression, associated with resistance to IMiDs. These data demonstrate that TNF-α secreted by BMSCs induces TRAF2 downregulation, thereby inducing IMiDs resistance. We further analyzed the mechanism of TNF-α-mediated TRAF2 downregulation. Since previous studies have shown that TRAF2 is a substrate of the proteasome, we treated MM cells with TNF-α in the presence and absence of proteasome inhibitor. TRAF2 downregulation by TNF-α was partially abrogated by proteasome inhibitor, associated with accumulation of ubiquitinated TRAF2. These data confirm that TNF-α induces TRAF2 downregulation is due, at least in part to proteasomal degradation. In conclusion, our data show that TRAF2 is a CRBN-independent regulator of IMiDs sensitivity and provide the preclinical rationale for combining IMiDs with inhibitors of non-canonical NF-κB or TNF-α signaling to overcome IMiDs resistance and improve patient outcome. Disclosures Anderson: Sanofi-Aventis: Other: Advisory Board; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/β complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

Activity of TAS4464, a novel NEDD8 activating enzyme E1 inhibitor, against multiple myeloma via inactivation of nuclear factor κB pathways.

The ubiquitin proteasome pathway is essential for the proliferation and survival of multiple myeloma (MM) cells. TAS4464, a novel highly potent inhibitor of NEDD8 activating enzyme, selectively inactivates cullin‐RING ubiquitin E3 ligases, resulting in accumulation of their substrates. Here, we examined 14 MM cell lines treated with TAS4464. TAS4464 induced growth arrest and cell death in the MM cell lines even in the presence of bone marrow stromal cells. It also induced the accumulation of phospho‐inhibitor of κBα and phospho‐p100, impaired the activities of nuclear factor κB (NF‐κB) transcription factors p65 and RelB, and decreased the expression of NF‐κB target genes, suggesting that TAS4464 inhibits both the canonical and non–canonical NF‐κB pathways. TAS4464 had similar effects in an in vivo human‐MM xenograft mouse model in which it was also observed to have strong antitumor effects. TAS4464 synergistically enhanced the antitumor activities of the standard MM chemotherapies bortezomib, lenalidomide/dexamethasone, daratumumab and elotuzumab. Together, these results suggest that the anti–MM activity of TAS4464 occurs via inhibition of the NF‐κB pathways, and that treatment with TAS4464 is a potential approach for treating MM by single and combination therapies.

The relationship between members of the canonical NF-kB pathway, tumour microenvironment and cancer specific survival in colorectal cancer patients.

The aim of this study was to investigate the role of the upstream kinase TAK1 and the canonical NF-κB pathway colorectal in cancer (CRC). Immunohistochemistry was used to assess the expression of TAK1/pTAK1 and canonical NF-κB pathway members in a tissue microarray of 242 patients. The relationship between expression, the tumour microenvironment and cancer-specific survival were examined. All the investigated members of the pathway were expressed in CRC tissue. In addition, cytoplasmic pTAK1 was associated with the tumour microenvironment (P=0.045) and cancer-specific survival (CSS) (P=0.032). When cytoplasmic pTAK1 was stratified by BRAF status, cytoplasmic pTAK1 expression association with CSS was strengthened (P=0.014). Cytoplasmic IKKβ was significantly associated with the inflammatory cell infiltrate (P=0.015) as graded by Klintrup Makinen grade, systemic inflammation as assessed by neutrophil-lymphocyte ratio (P=0.03) and CSS (P=0.046). On multivariate analysis cytoplasmic IKKβ was independently associated with CSS (HR 1.75,95%CI 1.05-2.91, P=0.033). Cytoplasmic pTAK1 was significantly associated with CSS and this was enhanced in patients with tumours that expressed wild type BRAF. High expression of cytoplasmic IKKβ was significantly associated with decreased CSS and with markers of the tumour microenvironment. These results support the hypothesis that NF-κB pathway members are poor prognostic markers in patients with CRC, but this requires to be validated in a large independent cohort.

Ex vivo infection of human placental explants with Trypanosoma cruzi and Toxoplasma gondii: Differential activation of NF kappa B signaling pathways

Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) are the causative agents of Chagas disease and Toxoplasmosis. T. cruzi and T. gondii present, respectively, low and high congenital transmission rates and induce a distinctive cytokine/chemokine profile in ex vivo infected human placental explants (HPE). Since the innate immune response is regulated, at least partially, by NF-κB signaling pathways, our main objective was to determine the effect of ex vivo infection with both parasites on the activation of canonical and non-canonical NF-κB pathways and its relation to parasite infection.T. cruzi activates both, the canonical and non-canonical pathways of NF-κB, unlike T. gondii, which has no effect on the canonical pathway and inhibits the non-canonical pathway. The inhibition of both pathways of NF-κB increases the DNA load of T. cruzi and T. gondii in HPE. Therefore, the differential modulation of NF-κB signal transduction pathways by both parasites might explain, at least partially, the low and high congenital transmission rates of T. cruzi and T. gondii.

Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays.

The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine.

TRAF6 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell expansion

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration, oxidative stress and pro-inflammatory cytokine production

Decursinol angelate (DA) is a pyranocoumarin purified from the roots of Angelica gigas. Here, we synthesized DA and determined its anti-inflammatory potential on TPA-induced mice ear inflammation. First, we evaluated the non-toxic behaviour of DA on HaCaT cells. Additionally, we observed the free radical scavenging potential of DA at 60 μM to be 50%. This finding was further supported by nitric oxide assay, malondialdehyde assay, H2DCFDA staining and western blotting analysis of antioxidant enzymes. DA also suppressed the activation and polarization of macrophage phagocytic activity on RAW 264.7 cells. We further evaluated the expression of ICAM-1, MCP-1, MIP-2 and MIP-1β on in-vivo model system. Consequently, DA significantly reduced the production of NF-κB and COX-2 induced proinflammatory cytokine levels on TPA induced ear edema. Inhibition of MAPK and transcriptional factor NF-κB was also validated by western blotting analysis of p-ERK, p-p38, IKKα, IKKγ, IκBα, NF-κB-p65. Immunohistochemistry and immunofluorescence staining of NFκB-p65, TNF-α and IL-1β were also performed to support the findings. Conclusively, these results suggest that topical administration of DA significantly inhibited the expression of pro-inflammatory cytokines by blocking the canonical NF-κB and MAPK pathway. Therefore, we suggest DA as a potent therapeutic compound against skin inflammation related diseases.

Airway M Cells Arise in the Lower Airway Due to RANKL Signaling and Reside in the Bronchiolar Epithelium Associated With iBALT in Murine Models of Respiratory Disease.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL–RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

RelB acts as a molecular switch driving chronic inflammation in glioblastoma multiforme

Glioblastoma multiforme (GBM) is a primary brain tumor characterized by extensive necrosis and immunosuppressive inflammation. The mechanisms by which this inflammation develops and persists in GBM remain elusive. We identified two cytokines interleukin-1β (IL-1) and oncostatin M (OSM) that strongly negatively correlate with patient survival. We found that these cytokines activate RelB/p50 complexes by a canonical NF-κB pathway, which surprisingly drives expression of proinflammatory cytokines in GBM cells, but leads to their inhibition in non-transformed astrocytes. We discovered that one allele of the gene encoding deacetylase Sirtuin 1 (SIRT1), needed for repression of cytokine genes, is deleted in 80% of GBM tumors. Furthermore, RelB specifically interacts with a transcription factor Yin Yang 1 (YY1) in GBM cells and activates GBM-specific gene expression programs. As a result, GBM cells continuously secrete proinflammatory cytokines and factors attracting/activating glioma-associated microglia/macrophages and thus, promote a feedforward inflammatory loop.

Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome

Inflammation in uveal melanoma (UM) is linked to a bad prognosis. It is rare type of cancer, of which the metastases are usually fatal within a year. Infiltration with an inflammatory infiltrate increases with disease progression but does not seem to inhibit metastasis. The Canonical NFκB (C-NFκB) pathway is known to play a crucial role in tumor inflammation. We therefore, studied the expression of canonical NFκB proteins and their prognostic relevance in UM. Our study evaluated the expression of C-NFκB proteins (p65, p50, and c-Rel) by using immunohistochemistry on sections from 75 formalin-fixed UM. Activation of the NFκB subunit was determined on fresh tumor specimens by measuring the DNA-binding activity in nuclei using an NFκB ELISA assay. Real-time PCR was performed on frozen material on 58 tumors. The presence of native C-NFκB heterodimers (p65/p50 and c-Rel/p50) was confirmed by co-immunoprecipitation followed by Western blotting. We observed a high nuclear immunoreactivity of p65, p50, and c-Rel proteins in 54, 60 and 41% UM cases, respectively. Expression of C-NFκB proteins significantly correlated with parameters which are related to the inflammatory environment of UM. Nuclear immunoreactivity of p65 and p50 was associated with lower patient survival (p = 0.041; p = 0.048) while c-Rel was not. Our finding reveals that C-NFκB proteins expressed are more often in UM with inflammation than those without inflammation. Activation of the canonical NFκB pathway is more frequent in high risk UM patients. These observations might help to understand the behaviour of high risk tumors, with upregulation of C-NFκB proteins contributing to tumor aggressiveness.

Correction: Dual Inhibition of Canonical and Noncanonical NF-κB Pathways Demonstrates Significant Antitumor Activities in Multiple Myeloma.

Correction: Dual Inhibition of Canonical and Noncanonical NF-κB Pathways Demonstrates Significant Antitumor Activities in Multiple Myeloma.