Lung cancer is a leading public health issue worldwide. Although therapeutic approaches have improved drastically in the last decades, the prognosis of lung cancer patients remains suboptimal. The canonical nuclear transcription factor kappa B (NF-κB) signalling pathway is critical in the carcinogenesis of lung cancer. The non-canonical NF-κB signalling pathway (represented by RelB) has attracted increasing attention in the pathogenesis of haematological and epithelial malignancies. However, the function of RelB in non-small cell lung cancer (NSCLC) is still unclear. Recently, high expression of RelB has been detected in NSCLC tissues. We have also demonstrated that RelB expression is an independent prognostic factor in NSCLC patients.

Lymphotoxin signaling via the lymphotoxin-beta receptor (LTbetaR) has been implicated in biological processes ranging from development of secondary lymphoid organs, maintenance of spleen architecture, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTbetaR crosslinking is NF-kappaB. Two signaling pathways have been described, the classical inhibitor of NF-kappaB alpha (IkappaBalpha)-regulated and the alternative p100-regulated pathway that result in the activation of p50-RelA and p52-RelB NF-kappaB heterodimers, respectively.

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-kappaB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFalpha) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFalpha synthesis in a manner that involved transcriptional repression of the TNFalpha promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFalpha promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFalpha cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFalpha production. Moreover, because Tat activates both RelB and TNFalpha in microglia, and because Tat induces inflammatory TNFalpha synthesis via NF-kappaB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-kappaB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND.

The NF-κB transcription factor regulates numerous immune responses but its contribution to interleukin-17 (IL-17) production by T cells is largely unknown. Here, we report that IL-17, but not interferon-γ (IFN-γ), production by γδ T cells required the NF-κB family members RelA and RelB as well as the lymphotoxin-β-receptor (LTβR). In contrast, LTβR-NF-κB signaling was not involved in the differentiation of conventional αβ Th17 cells. Impaired IL-17 production in RelA- or RelB-deficient T cells resulted in a diminished innate immune response to Escherichia coli infection. RelA controlled the expression of LT ligands in accessory thymocytes whereas RelB, acting downstream of LTβR, was required for the expression of RORγt and RORα4 transcription factors and the differentiation of thymic precursors into γδT17 cells. Thus, RelA and RelB within different thymocyte subpopulations cooperate in the regulation of IL-17 production by γδ T cells and contribute to the host's ability to fight bacterial infections.

Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.

Laryngeal cancer is a frequent malignancy originating from the squamous vocal epithelium in a multi-stage fashion in response to environmental carcinogens. Although most cases can be cured by surgery and/or radiotherapy, advanced and relapsing disease is common, and biomarkers of such dismal cases are urgently needed. The cancer genome of laryngeal cancers was recently shown to feature a signature of aberrant nuclear factor (NF)-κB activation, but this finding has not been clinically exploited. We analyzed primary tumor samples of 96 well-documented and longitudinally followed patients covering the whole spectrum of laryngeal neoplasia, including 21 patients with benign laryngeal diseases, 15 patients with dysplasia, 43 patients with early-stage carcinoma, and 17 patients with locally advanced carcinoma, for immunoreactivity of RelA, RelB, P50, and P52/P100, the main NF-κB subunits that activate transcription. Results were cross-examined with indices of tumor progression and survival. Interestingly, RelB expression increased with tumor stage, grade, and local extent. Moreover, patients displaying high RelB immunoreactivity exhibited statistically significantly poorer survival compared with patients featuring low levels of RelB expression (P = 0.018 by log-rank test). Using Cox regression analyses and tumor stage, local extent, grade and RelA/RelB immunoreactivity, we develop a new score that can independently predict survival of patients with laryngeal cancer. Hence we provide a simple and affordable NF-κB-based test to predict prognosis in laryngeal cancer.

The interaction between programmed death receptor (PD-1) and its ligand (PD-L1) is essential for suppressing activated T-lymphocytes. However, the precise mechanisms underlying PD-L1 overexpression in tumours have yet to be fully elucidated. Here, we describe that RelB participates in the immune evasion of prostate cancer (PCa) via cis/trans transcriptional upregulation of PD-L1.

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway. Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs. Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation. Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

Tamoxifen (TAM) resistance remains a major obstacle in the treatment of advanced breast cancer (BCa). In addition to the competitive inhibition of the estrogen receptor (ER) signaling pathway, damping of mitochondrial function by increasing reactive oxygen species (ROS) is critical for enhancing TAM pharmacodynamics. Here, we showed that RelB contributes to TAM resistance by inhibiting TAM-provoked ferroptosis. TAM-induced ROS level promoted ferroptosis in TAM-sensitive cells, but the effect was alleviated in TAM-resistant cells with high constitutive levels of RelB. Mechanistically, RelB inhibited ferroptosis by transcriptional upregulating glutathione peroxidase 4 (GPX4). Consequently, elevating RelB and GPX4 in sensitive cells increased TAM resistance, and conversely, depriving RelB and GPX4 in resistant cells decreased TAM resistance. Furthermore, suppression of RelB transcriptional activation resensitized TAM-resistant cells by enhancing ferroptosis in vitro and in vivo. The inactivation of GPX4 in TAM-resistant cells consistently resensitized TAM by increasing ferroptosis-mediated cell death. Together, this study uncovered that inhibition of ferroptosis contributes to TAM resistance of BCa via RelB-upregulated GPX4.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB-/- mice, which have spontaneous multiorgan autoimmune disease. RelB-/- thymi were organized, with medullary structures containing AIRE- mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB-/- thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). It is firmly established that overactivation of the p65 (RelA) nuclear factor kappa B (NF-κB) transcription factor upregulates expression of inflammatory mediators in both immune and non-immune resident CNS cells and promotes inflammation during MS. In contrast to p65, NF-κB family member RelB regulates immune cell development and can limit inflammation. Although RelB expression is induced during inflammation in the CNS, its role in MS remains unknown.

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

Liver fibrosis is a serious chronic disease that developed by a coordinated interplay of many cell types, but the underlying signal transduction in individual cell type remains to be characterized. Nuclear factor-κB (NF-κB) is a widely accepted central player in the development of hepatic fibrosis. However, the precise role of each member of NF-κB in different cell type is unclear. Here, we generated a mouse model (RelbΔhep ) with hepatocyte-specific deletion of RelB, a member of NF-κB family. RelbΔhep mice born normally and appear normal without obvious abnormality. However, in the CCl4-induced liver fibrosis, RelbΔhep mice developed less severe disease compared with wide-type (WT) mice. The denaturation and necrosis of hepatocytes as well as the formation of false lobules in RelbΔhep mice were significantly reduced compared with WT mice. The production of α-SMA and the level of collagen I and Collagen III were greatly reduced in RelbΔhep mice comparing with WT mice. Furthermore, in patients with liver fibrosis, RelB is up-regulated along with the stage of diseases. Consistently, CCl4 treatment could up-regulate the expression of RelB as well as inflammatory cytokines such as IL-6 and TGF-β1 in hepatoma cell as well as in WT mice. Knockdown the expression of RelB in hepatoma cells greatly reduced the expression of CCl4-induced inflammatory cytokines. In summary, we provide the genetic evidence to demonstrate the critical and hepatocellular role of RelB in liver fibrosis. RelB is an important transcription factor to drive the expression of inflammatory cytokines in the initiation phase of injury.

NF-kappaB (NF-κB) is a family of transcription factors with pleiotropic functions in immune responses. The alternative NF-κB pathway that leads to the activation of RelB and NF-κB2, was previously associated with the activation and function of T cells, though the exact contribution of these NF-κB subunits remains unclear. Here, using mice carrying conditional ablation of RelB in T cells, we evaluated its role in the development of conventional CD4+ T (Tconv) cells and their function in autoimmune diseases. RelB was largely dispensable for Tconv cell homeostasis, activation and proliferation, and for their polarization toward different flavors of Thelper cells in vitro. Moreover, ablation of RelB had no impact on the capacity of Tconv cells to induce autoimmune colitis. Conversely, clinical severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS) was significantly reduced in mice with RelB-deficient T cells. This was associated with impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically in the central nervous system. Our data reveal a discrete role for RelB in the pathogenic function of Tconv cells during EAE, and highlight this transcription factor as a putative therapeutic target in MS.

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.

Nuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemo-sensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. However, the significance of RelB in colorectal cancer (CRC) remains unclear. Here, we systematically explored the functions of the alternative NF-κB subunit RelB in colon cancer cells and its underlying mechanism.

The development of radioresistance is one of main causes for therapeutic failure of prostate cancer (PCa). The present study aims to investigate the function and the related mechanism by which HZ08 sensitizes radiotherapeutic efficiency to treat aggressive PCa cells.

Sepsis is more common among males than females, and the unequal estrogen levels have been suspected to play a vital role in gender differences. Recently, trained immunity is reported to be a novel strategy for the innate immune system to fight infection. However, it has not been clarified whether β-glucan-induced trained immunity causes different responses to early sepsis between male and female mice. In this study, sepsis was induced in mice by intraperitoneal injection of Escherichia coli (E. coli). The changes of inflammatory cytokines expression, and macrophage polarization in male, female, and ovariectomized C57BL/6 mice in sepsis model were investigated. For in vitro studies, different macrophages were treated with LPS. The function of estradiol (E2) on macrophage cell lines was verified and the mechanism of E2 affecting trained immunity was explored. We demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. Macrophage polarization toward the M1 phenotype, which exhibited enhanced trained immunity, was related to the difference in sepsis resistance between female and male mice. Moreover, ovariectomized (OVX) mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. Female bone marrow-derived macrophages (BMDMs) were also apt to be polarized to the M1 phenotype in response to trained immunity in vitro. Furthermore, E2 promoted trained immunity in macrophage cell lines J774 and RAW264.7. E2 was also verified to facilitate trained immunity in primary BMDMs from female and male mice. Mechanistically, we found that E2 inhibited the nuclear translocation of RelB, which is a member of non-canonical pathway of NFκB and contributes to macrophage polarization to change the intensity of trained immunity. This study is the first to indicate the role of E2 in the trained immunity induced by β-glucan to protect against E. coli-induced sepsis via the non-canonical NFκB pathway. These results improve our understanding of the molecular mechanisms governing trained immunity in gender differences.

Activation of CD40-signaling contributes to the initiation, progression and drug resistance of B cell lymphomas. We contributed to this knowledge by showing that constitutive CD40-signaling in B cells induces B cell hyperplasia and finally B cell lymphoma development in transgenic mice. CD40 activates, among others, the non-canonical NF-ĸB signaling, which is constitutively activated in several human B cell lymphomas and is therefore presumed to contribute to lymphopathogenesis. This prompted us to study the regulatory role of the non-canonical NF-ĸB transcription factor RelB in lymphomagenesis. To this end, we crossed mice expressing a constitutively active CD40 receptor in B cells with conditional RelB-KO mice. Ablation of RelB attenuated pre-malignant B cell expansion, and resulted in an impaired survival and activation of long-term CD40-stimulated B cells. Furthermore, we found that hyperactivation of non-canonical NF-кB signaling enhances the retention of B cells in the follicles of secondary lymphoid organs. RNA-Seq-analysis revealed that several genes involved in B-cell migration, survival, proliferation and cytokine signaling govern the transcriptional differences modulated by the ablation of RelB in long-term CD40-stimulated B cells. Inactivation of RelB did not abrogate lymphoma development. However, lymphomas occurred with a lower incidence and had a longer latency period. In summary, our data suggest that RelB, although it is not strictly required for malignant transformation, accelerates the lymphomagenesis of long-term CD40-stimulated B cells by regulating genes involved in migration, survival and cytokine signaling.

Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3'-UTR AREs. We have recently reported that TTP preferentially up-regulates the expression level of innate immunity genes involved in the type I interferon-mediated signaling pathway and viral response in cancer cells. To elucidate the role of TTP-RNA interaction in TTP-mediated upregulation of gene expression, we performed iRIP-seq experiments to obtain the RNA interaction map consisting of direct and indirect binding sites of TTP in HeLa cells. We found substantial TTP binding signals in mRNA regions and the introns. ARE-motif AUUUA is over-represented in TTP binding peaks. Strikingly, AUUUA frequency is high both in 3'UTR and intronic regions, and the intronic peaks were more associated with TTP-regulated genes. Analysis of the over-represented motifs in TTP peaks revealed the high frequencies of UAGG and GUGUG motifs reported for hnRNPA2/B1 and CELF1 respectively in the 3'UTR and introns, and also the UGGAC motif overlapping with the m6A motif GGACU in the CDS regions. We further demonstrated that TTP binds to multiple intronic and exonic sites in the pre-mRNA/mRNA of the transcription factor RelB, correlating with the TTP-upregulated expression of RelB. TTP-up-regulated genes without a TTP binding site, but not those with, are highly enriched in innate immunity pathways and show higher tendency of harboring RelB binding sites in their promoter regions. These findings support a model in which TTP binding of RelB pre-mRNA/mRNA coordinates the RelB upregulation and activation of the innate immunity for antiviral response.