Negative Regulator Of NF-κB Signaling Research Articles

TAX1BP1/A20 inhibited TLR2-NF-κB activation to induce tolerant expression of IL-6 in endothelial cells

The inflammatory cascadedriven by interleukin-6 (IL-6) plays a crucial role in the initiation and progression of chronic inflammatory conditions such as atherosclerosis. Research has demonstrated that prolonged exposure to inflammatory stimuli leads to the development of “immune tolerance” in specialized immune cells such as monocytes and macrophages, serving as a mechanism to prevent tissue damage and curb the inflammatory cascade. However, our recent investigation revealed that immune tolerance did not effectively regulate the production of IL-6 in human umbilical vein endothelial cells (HUVECs) when stimulated by a Toll-like receptor 2 (TLR2) ligand Pam3CSK4, which is a potent activator of the pro-inflammatory transcription factor NF-κB. Furthermore, the negative regulator of NF-κB signaling, A20, was ineffective in suppressing TLR2-induced IL-6 synthesis in this context. Notably, all A20 auxiliary molecules, with the exception of TAX1BP1, were found to be significantly expressed in HUVECs. DNA methylation in TAX1BP1 was confirmed in GEO database. According to the information provided, it is hypothesized that altered DNA methylation in HUVECs could potentially lead to decreased expression of TAX1BP1, thereby impeding A20′s capacity to modulate continuous activation of the TLR2-NF-κB pathway. This may consequently lead to unregulated production of IL-6, evading immune tolerance mechanisms. Subsequent investigations suggested that demethylating TAX1BP1 could enhance its expression, potentially reducing the endogenous IL-6 levels induced by repeated TLR2 stimulation and restoring A20′s inhibitory role in NF-κB signaling. Additionally, over-expression of TAX1BP1 coulddecrease the production of atherosclerosis-associated cytokines like IL-6, MCP-1, ICAM-1, and VCAM-1, while increasing NO release following repeated Pam3cks4 stimulation, along with enhanced co-localization of TAX1BP1 and A20. These findings indicate that inducing immune tolerance in endothelial cells may effectively suppress endogenous IL-6 production and halt the IL-6-mediated inflammatory cascade, with TAX1BP1/A20 identified as crucial components in this process.These insights provide novel perspectives and potential targets for therapeutic strategies in inflammatoryimmunological disorders involving the overproduction of IL-6.

TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Ammonia stress-induced heat shock factor 1 enhances white spot syndrome virus infection by targeting the interferon-like system in shrimp.

Ammonia is the end product of protein catabolism and is derived from feces and unconsumed foods. It threatens the health and growth of aquatic animals. In this study, we demonstrated that ammonia stress suppresses shrimp antiviral immunity by targeting the shrimp interferon-like system and that heat shock factor 1 (Hsf1) is a central regulator of this process. When shrimp are stressed by ammonia, they activate Hsf1 for stress relief and well-being. Hsf1 upregulates the expression of negative regulators that inhibit the production and function of interferon analogs in shrimp, thereby enhancing white spot syndrome viral infection. Therefore, this study, from a molecular perspective, explains the problem in the aquaculture industry that animals living in stressed environments are more susceptible to pathogens than those living in unstressed conditions. Moreover, this study provides new insights into the side effects of heat shock responses and highlights the complexity of achieving cellular homeostasis under stressful conditions.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of β-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with β-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to β-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1β, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides β-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. Oral epithelial cells express Dectin-1 and recognize β-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1β from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1β result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

GRK2, an Inhibitor of MALT1-Dependent Oncogenic Signaling, Is Downregulated By microRNA in ABC-DLBCL

Background: The “CBM” complex, composed of the scaffolding protein CARMA1, the adaptor protein BCL10, and the effector protein MALT1, performs multiple pivotal functions as a mediator of antigen receptor-dependent induction of the NF-κB transcription factor and subsequent lymphocyte activation. The effector molecule of the CBM complex, MALT1, regulates downstream signaling via two important functions: first, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of NF-κB signaling. Deregulated/constitutive activation of MALT1, which can result from gain-of-function mutations in either B cell receptor (BCR) or CARMA1 or from chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of a variety of lymphoid malignancies including activated B-cell type-diffuse large B-cell lymphoma (ABC-DLBCL) and mucosa associated lymphoid tissue (MALT) lymphoma. We recently discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1 and inhibits MALT1 scaffold and proteolytic activities. Further, we found that knockdown of GRK2 in ABC-DLBCL enhances tumor growth in vivo, suggesting that GRK2 may act as a tumor suppressor in MALT1-dependent lymphomas. Interestingly, we found that GRK2 mRNA levels are markedly lower in a subset of DLBCL cases in comparison to normal B cell controls and that lower GRK2 level is associated with reduced survival in ABC-DLBCL patients. We thus sought to investigate how GRK2 expression is regulated in ABC-DLBCL. Methods and Results: We surveyed 6 published DLBCL sequencing datasets (334 cases total) and identified only one case with a mutation/deletion in the ADRBK1 (GRK2) gene. Thus, we concluded that mutation/deletion could not account for the lower levels of GRK2 in a subset of DLBCL cases. We next investigated other potential mechanisms that could play a role in downregulating GRK2 in ABC-DLBCL, including the impact of microRNA (miRNA). Using DICER-knock-down (KD) and knock-out (KO) HEK 293T cells, we demonstrated that GRK2 protein level significantly increases when miRNA processing is impaired. Next, we performed a bioinformatic analysis which identified two candidate microRNAs, miR-148b and miR-331, whose levels inversely correlate with GRK2 mRNA in ABC-DLBCL patient tumor specimens. These two microRNAs were also identified among the top four candidate hits (miR-125b, miR-148b, miR-125a and miR-331) using mirDIP, a microRNA Data Integration Portal which we used to screen for candidate miRNAs predicted to target the 3'UTR of GRK2. We confirmed direct targeting of GRK2 by three top candidate miRNAs, miR-148b, miR-125a and miR-125b, using a GRK2 3'UTR reporter assay. Notably, we found that expression of these three microRNAs is higher in ABC-DLBCL cells (OCI-Ly3 and TMD8), which demonstrate lower GRK2 mRNA expression in comparison to primary B cells. We showed that overexpression of either miR-125a, miR-125b or miR-148 in OCI-Ly3 and TMD8 cells leads to reduced GRK2 protein level, increased MALT1 scaffolding and proteolytic activities and enhanced ABC-DLBCL cell proliferation. Conversely, anti-miRNA Locked Nucleic Acids (LNA) inhibitors of miR-125a, miR-125b or miR-148 each inhibited ABC-DLBCL cell proliferation and MALT1 activity. Further evaluation of the effect of anti-miRNA LNA inhibitors on tumor progression is under way, using an ABC-DLBCL xenograft animal model. Conclusions: Together, our data show that specific miRNAs (miR-148b, miR-125a and miR-125b) down-regulate GRK2 expression in ABC-DLBCL, which in turn enhances MALT1 scaffolding and proteolytic activities, leading to increased tumor cell proliferation. Future studies investigating miRNA inhibitors which enhance GRK2 expression and thereby suppress MALT1 activity may inform novel strategies for inhibiting MALT1-dependent lymphomagenesis.

ANGI-04. IKIP SUPPRESSES MIGRATION AND INVASION OF GLIOBLASTOMA BY DOWNREGULATING THBS1/FAK SIGNALING

Abstract BACKGROUND The role of the inhibitor of NF-κB kinase-interacting protein (IKIP) in glioblastoma (GBM) migration and invasion remains poorly understood, despite its known involvement in the negative regulation of NF-κB signaling and inflammation. This study aimed to investigate the impact of IKIP on GBM metastasis and elucidate the underlying molecular mechanisms. METHODS Expression levels of IKIP were analyzed in gliomas and normal brain tissues using bioinformatic analysis of public databases and immunohistochemical staining of clinical samples. Transwell and wound healing assays were employed to assess the migration and invasion of GBM cell lines. RNA sequencing was conducted to unravel the molecular pathways involved. RESULTS Bioinformatic analysis revealed a significantly higher expression of IKIP in GBM compared to normal brain tissues, and its upregulation was associated with a poor prognosis in glioma patients. However, immunohistochemical and immunofluorescent staining demonstrated the downregulation of IKIP in GBM. Furthermore, functional assays demonstrated that IKIP overexpression suppressed the migratory and invasive capabilities of GBM cell lines, whereas IKIP knockdown enhanced migration and invasion. Mechanistically, IKIP overexpression downregulated THBS1 at the transcriptional level, resulting in the inhibition of THBS1/FAK signaling. CONCLUSIONS This study provides evidence for the first time that IKIP exerts inhibitory effects on GBM migration and invasion by downregulating THBS1/FAK signaling. These findings shed light on the complex mechanisms underlying GBM progression and identify IKIP as a potential therapeutic target for GBM invasion.

MiR-196b-5p activates NF-κB signaling in non-small cell lung cancer by directly targeting NFKBIA

BackgroundOur recent study found that QKI-5 regulated miRNA, miR-196b-5p, promotes non-small cell lung cancer (NSCLC) progression by directly targeting GATA6, TSPAN12 and FAS. However, the biological functions of miR-196b-5p in NSCLC progression and metastasis still remain elusive. MethodsCell proliferation, migration, colony formation, cell cycle assays were used to investigate cellular phenotypic changes. Quantitative real-time PCR (qRT-PCR) and western blot analyses were used to measure expressions of relative gene and protein. Interaction between QKI-5 and miR-196b-5p was determined by RNA immunoprecipitation (RIP) assay. Luciferase reporter assay was used to determine direct binding between miR-196b-5p and NFKBIA 3′-UTR. ELISA assay was used to measure secreted IL6 proteins. Mice xenograft model was used to assess the functions of NFKBIA on in vivo tumor growth. ResultsWe demonstrated that the miR-196b-5p facilitates lung cancer cell proliferation, migration, colony formation, and cell cycle by directly targeting NFKBIA, a negative regulator of NFκB signaling. Knocking down NFKBIA increases IL6 mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote lung cancer cell growth by activating NFκB signaling. The expression of NFKBIA was significantly downregulated in NSCLC tissue samples, and was negatively correlated with the expression miR-196b-5p. In addition, we found that downregulated QKI-5 expression was associated with the elevated miR-224 expression in NSCLC. ConclusionsOur findings indicated that the miR-224/QKI-5/miR-196b-5p/NFKBIA signaling pathway might play important functions in the progression of NSCLC, and suggested that targeting this pathway might be an effective therapeutic strategy in treating NSCLC.

Co-Targeting Nucleus Accumbens Associate 1 and NF-κB Signaling Synergistically Inhibits Melanoma Growth.

Nucleus-accumbens-associated protein-1 (NAC1) is a cancer-related transcriptional factor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and has been appreciated as one of the top potential cancer driver genes. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. Here, we show that NAC1 is a negative regulator of NF-κB signaling, and NAC1 depletion enhances the level of the nuclear NF-κB in human melanoma. Furthermore, the inhibition of NF-κB signaling significantly potentiates the antineoplastic activity of the NAC1 inhibition in both the cultured melanoma cells and xenograft tumors. This study identifies a novel NAC1-NF-κB signaling axis in melanoma, offering a promising new therapeutic option to treat melanoma.

TLR5S negatively regulates the TLR5M-mediated NF-κB signaling pathway in Epinephelus coioides

Nuclear factor kappa-B (NF-κB) pathway is a key mediator of inflammation response that plays a role in host defense for pathogen elimination, but excessive activation may lead to tissue damage or pathogen transmission. The negative regulation of NF-κB in lower vertebrates is largely unknown, hindering further understanding of immune signaling evolution. Here, we provided evidence that Epinephelus coioides soluble toll-like receptor 5 (TLR5S), a member of the TLR5 subfamily, has been newly identified as a negative regulator of NF-κB signaling. EcTLR5S was a cytoplasmic protein consisting of 17 leucine-rich repeat domains, which specifically responded to Vibrio flagellin and suppressed flagellin-induced NF-κB signaling activation and cytokine expression. The amino-terminal LRR 1–5 region was necessary for its negative regulatory function. Dual-luciferase reporter assay showed that EcTLR5S significantly inhibited the NF-κB-luc activity induced by inhibitor of NF-κB kinase α (IKKα) and IKKβ. Subsequently, the functional relationship between EcTLR5M and EcTLR5S was analyzed, revealing that the negative regulatory function of EcTLR5S targeted the activation of the NF-κB pathway mediated by EcTLR5M. The above results reveal that EcTLR5S negatively regulates the flagellin-induced EcTLR5M-NF-κB pathway activation, which may prevent over-activation of immune signaling and restore homeostasis.

YTHDF2 inhibition potentiates radiotherapy antitumor efficacy.

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression. However, the impact of m6A on the antitumor effects of radiotherapy and the related mechanisms are unknown. Here we show that ionizing radiation (IR) induces immunosuppressive myeloid-derived suppressor cell (MDSC) expansion and YTHDF2 expression in both murine models and humans. Following IR, loss of Ythdf2 in myeloid cells augments antitumor immunity and overcomes tumor radioresistance by altering MDSC differentiation and inhibiting MDSC infiltration and suppressive function. The remodeling of the landscape of MDSC populations by local IR is reversed by Ythdf2 deficiency. IR-induced YTHDF2 expression relies on NF-κB signaling; YTHDF2 in turn leads to NF-κB activation by directly binding and degrading transcripts encoding negative regulators of NF-κB signaling, resulting in an IR-YTHDF2-NF-κB circuit. Pharmacological inhibition of YTHDF2 overcomes MDSC-induced immunosuppression and improves combined IR and/or anti-PD-L1 treatment. Thus, YTHDF2 is a promising target to improve radiotherapy (RT) and RT/immunotherapy combinations.

Protective effect of TNIP2 on the inflammatory response of microglia after spinal cord injury in rats

BackgroundSpinal cord injury (SCI) is a devastating disease that can lead to tissue loss and neurological dysfunction. TNIP2 is a negative regulator of NF-κB signaling due to its capacity to bind A20 and suppress inflammatory cytokines-induced NF-κB activation. However, the anti-inflammatory role of TNIP2 in SCI remains unclear. Our study's intention was to evaluate the effect of TNIP2 on the inflammatory response of microglia after spinal cord injury in rats. MethodsHE staining and Nissl staining were performed on day 3 following SCI to analyze the histological changes. To further investigate the functional changes of TNIP2 after SCI, we performed immunofluorescence staining experiments. The effect of LPS on TNIP2 expression in BV2 cells was examined by western blot. The levels of TNF-α, IL-1β, and IL-6 in spinal cord tissues of rats with SCI and in BV2 cells with LPS were measured by using qPCR. ResultsTNIP2 expression was closely associated with the pathophysiology of SCI in rats, and TNIP2 was involved in regulating functional changes in microglia. TNIP2 expression was increased during SCI in rats and that overexpression of TNIP2 inhibited M1 polarization and pro-inflammatory cytokine production in microglia, which might ultimately protect against inflammatory responses through the MAPK and NF-κB signaling pathways. ConclusionsThe present study provides evidence for a role of TNIP2 in the regulation of inflammation in SCI and suggests that induction of TNIP2 expression alleviated the inflammatory response of microglia.

TNIP2 inhibits amyloidogenesis by regulating the 3′UTR of BACE1: An in vitro study

TNFAIP3-interacting protein 2 (TNIP2) is known as a negative regulator of NF-κB signaling and inhibit inflammatory response and apoptosis, and is also involved in RNA metabolism. In this study, we investigated the potential role of TNIP2 in amyloidogenesis critically associated with Alzheimer’s disease (AD). We found a significant decline of TNIP2 protein level in both mouse and cell model of AD. In SH-SY5Y and HEK cells that stably express human full-length APP695 (SY5Y-APP and HEK-APP), TNIP2 overexpression decreased the protein levels of β-secretase (BACE1) and C99, as well as Aβ peptides (including Aβ40 and Aβ42), while those of α-secretase (ADAM10) and the related C83 remained unchanged. We further found that TNIP2 promoted the degradation of BACE1 mRNA and was able to bound to the 3′ untranslated region (3′UTR) with the reduced luciferase activity. These results indicated that TNIP2 effectively inhibited amyloidogenic processing by regulating the 3′UTR-associated mRNA decay of BACE1.

HBV X Protein Induces Degradation of UBXN7, a Novel Negative Regulator of NF-κB Signaling, to Promote HBV Replication.

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using invitro and invivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase β through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.

ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression

Chronic inflammation and an immunosuppressive microenvironment promote prostate cancer (PCa) progression and diminish the response to immune checkpoint blockade (ICB) therapies. However, it remains unclear how and to what extent these two events are coordinated. Here, we show that ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, functions downstream of inflammation-induced IKKβ activation to shape the immunosuppressive tumor microenvironment (TME). Prostate-specific deletion of Arid1a cooperates with Pten loss to accelerate prostate tumorigenesis. We identify polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) as the major infiltrating immune cell type that causes immune evasion and reveal that neutralization of PMN-MDSCs restricts the progression of Arid1a-deficient tumors. Mechanistically, inflammatory cues activate IKKβ to phosphorylate ARID1A, leading to its degradation via β-TRCP. ARID1A downregulation in turn silences the enhancer of A20 deubiquitinase, a critical negative regulator of NF-κB signaling, and thereby unleashes CXCR2 ligand-mediated MDSC chemotaxis. Importantly, our results support the therapeutic strategy of anti-NF-κB antibody or targeting CXCR2 combined with ICB for advanced PCa. Together, our findings highlight that the IKKβ/ARID1A/NF-κB feedback axis integrates inflammation and immunosuppression to promote PCa progression.

USP14-mediated NLRC5 upregulation inhibits endothelial cell activation and inflammation in atherosclerosis

Atherosclerosis, a chronic inflammatory condition that leads to a variety of life-threatening cardiovascular diseases, is a worldwide public health concern. Endothelial cells (ECs), which line the inside of blood vessels, play an important role in atherogenic initiation. Endothelial activation and inflammation are indispensable for the early stage of atherosclerosis. Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme that regulates the stability and activity of target proteins, has been identified as a potential therapeutic target for many inflammatory diseases. However, the role of USP14 on ECs is undefined. In this study, we found that USP14 is downregulated in either atherosclerosis patient specimens or oxidized low-density lipoprotein (ox-LDL)-stimulated ECs as compared to the control group. Overexpression of USP14 in ECs restrains ox-LDL-stimulated nuclear transcription factor kappa B (NF-κB) activation and subsequent adhesion molecule production. USP14 inhibits endothelium proinflammatory activation by suppressing the degradation of the negative regulator of NF-κB signaling, nod-like receptor family caspase recruitment domain family domain containing 5 (NLRC5). Finally, our in vivo experiments confirmed that USP14 adenovirus injection in apolipoprotein E deficient (ApoE−/−) mice fed with a western diet reduced the atherosclerotic lesion size, inhibited macrophage accumulation in the intima, and restricted the progression of atherosclerosis. Our results reveal that USP14 may represent a new therapeutic target for atherosclerosis.

Epigenetic Silencing of Tumor Suppressor lncRNA NKILA: Implication on NF-κB Signaling in Non-Hodgkin's Lymphoma.

The long non-coding RNA (lncRNA) NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling implicated in carcinogenesis. As a CpG island is embedded in the promoter region of NKILA, it is hypothesized as a tumor suppressor lncRNA silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). By pyrosequencing-verified methylation-specific PCR, NKILA methylation was detected in 1/10 (10%) NHL cell lines, but not in normal peripheral blood buffy coats or tonsils. NKILA methylation correlated with the repression of NKILA in cell lines. Hypomethylation treatment with 5-Aza-2′-deoxycytidine resulted in promoter demethylation and the re-expression of NKILA. In 102 NHL primary samples, NKILA was methylated in 29 (51.79%) diffuse large B-cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma cases, but unmethylated in all 26 mantle cell lymphoma cases. Mechanistically, the knockdown of NKILA resulted in promoting IkBα phosphorylation, associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, the knockdown of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation. Collectively, NKILA was a tumor suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing resulted in increased cellular proliferation and decreased cell death via the repression of NF-κB signaling in NHL.

Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

Spata2 Knockdown Exacerbates Brain Inflammation via NF-κB/P38MAPK Signaling and NLRP3 Inflammasome Activation in Cerebral Ischemia/Reperfusion Rats.

Brain inflammation induced by ischemic stroke is an important cause of secondary brain injury. The nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and NLRP3 inflammasome signaling are believed to drive the progression of brain inflammation. Spermatogenesis-associated protein2 (SPATA2) functions as a partner protein that recruits CYLD, a negative regulator of NF-κB signaling, to signaling complexes. However, the role of SPATA2 in the central nervous system remains unclear and whether it is involved in regulating inflammatory responses remains controversial. Rats were subjected to transient middle cerebral artery occlusion followed by reperfusion (tMCAO/R) surgery. The expression and localization of SPATA2 in the brain were investigated. The lentivirus-mediated shRNA was employed to inhibit SPATA2 expression. The inflammatory responses and outcomes of Spata2 knockdown were investigated. SPATA2 was co-localized with CYLD in neurons. SPATA2 expression was reduced in tMCAO/R rats. Spata2 knockdown resulted in increased microglia, increased expression of Tnfa, Il-1β, and Il-18, decreased Garcia score, and increased infarct volume. Spata2 knockdown resulted in the activation of P38MAPK and NLRP3 inflammasome and the increased activation of NF-κB signaling. These results suggest that SPATA2 plays a protective role against brain inflammation induced by ischemia/reperfusion injury. Therefore, SPATA2 could be a potential therapeutic target for treating ischemic stroke.

Endothelial ERG determines the Immune Niche and Vascular Homeostasis

Increased vascular permeability and uncontrolled accumulation of activated neutrophils in the interstitium is the hall mark of acute lung injury. The vascular endothelium, which lines all blood vessels, is the first entry point for neutrophils transmigration to underlying tissue. In general, naïve endothelium is “unactivated or anti-inflammatory” as it dynamically allows neutrophils to survey the underlying tissue for host-defense. How then endothelium acquire “immune activated state” without encountering infectious injury leading to neutrophils influx and inflammatory tissue injury remains elusive. We have discovered that ERG, a transcription factor belonging to ETS related gene (ERG), instructs endothelial anti-inflammatory niche and thereby prevents neutrophil activation and vascular injury. We found that conditional deletion of ERG in adult endothelium of the mice (iEC-ERG-/-mice) spontaneously increased vascular permeability and accumulation of activated neutrophils in the air space. Moreover, iEC-ERG-/- mice developed inflammation as reflected by increased NFκB activity and pro-inflammatory cytokines/chemokines accumulation including IL-8. Neutrophil egress is influenced by the local production of chemokine ligands like IL-8. Moreover, deubiquitinase A20 is the critical negative regulator of NFκB signalling and thereby inflammation. We show that loss of ERG in endothelium led to reduced A20 expression in mouse and human lung endothelial cells (EC). We therefore assess if rescuing A20 levels or inhibiting IL8 will restore lung inflammatory injury in EC-ERG null mice. Rescuing A20 expression in human lung EC suppressed the level of cytokines to the level as seen in controls. To investigate the role of IL-8 in activating neutrophil influx we used Reparixin, an allosteric inhibitor of IL-8 receptor. Reparixin was administered to iEC-ERG-/-mice and FACS analysis was performed. Results showed markedly reduced inflammation and neutrophil influx in the Reparixin treated ERG null mice. Collectively, our observations show that ERG depletion causes low A20 levels leading to uncontrolled NFκB activation and IL-8 chemokine secretions triggering neutrophil mobilization. We conclude that ERG expression is required in endothelium for maintaining anti-inflammatory ‘immune niche’ to prevent neutrophil influx and inflammatory injury.