Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Non-canonical NF-κB signaling limits the tolerogenic ß-catenin-Raldh2 axis in gut dendritic cells to exacerbate intestinal pathologies.

Dendritic cell (DC) dysfunction is known to exacerbate intestinal pathologies, but the mechanisms compromising DC-mediated immune regulation in this context remain unclear. Here, we show that intestinal dendritic cells from a mouse model of experimental colitis exhibit significant levels of noncanonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviates intestinal pathologies in mice suffering from colitis. Deficiency of RelB:p52 diminishes transcription of Axin1, a critical component of the ß-catenin destruction complex, reinforcing ß-catenin-dependent expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid synthesis. DC-specific impairment of noncanonical NF-κB signaling leads to increased colonic numbers of Tregs and IgA+ B cells, which promote luminal IgA production and foster eubiosis. Experimentally introduced ß-catenin haploinsufficiency in DCs with deficient noncanonical NF-κB signaling moderates Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, inflammatory bowel-disease patients also display a deleterious noncanonical NF-κB signaling signature in intestinal DCs. In sum, we establish how noncanonical NF-κB signaling in dendritic cells can subvert retinoic acid synthesis to fuel intestinal inflammation.

Transcriptional factor BRD4 promotes the stemness of esophageal cancer by activating the nuclear PD-L1/RelB axis.

Esophageal cancer (EC) is a prevalent malignancy associated with therapeutic resistance and poor prognosis. This study investigates the role of programmed death-ligand 1 (PD-L1) in esophageal cancer stem cell (ECSC) formation. ECSCs were enriched and characterized using various assays. We found that both PD-L1 and bromodomain-containing protein 4 (BRD4) were upregulated in ECSCs, promoting their stemness. Inhibiting BRD4 suppressed ECSC markers expression and sphere formation. Furthermore, BRD4 inhibitors downregulated membrane and nuclear PD-L1 levels, with knockdown of PD-L1 inhibiting ECSC formation. PD-L1 degraders also affected PD-L1 and its downstream effector RelB expression. Moreover, inhibiting RelB influenced sphere formation through interleukin-6 expression. This study reveals the critical role of the BRD4/nuclear PD-L1/RelB axis in ECSC formation, highlighting nuclear PD-L1 as a potential immunotherapeutic target for refractory EC.

RelB-deficient autoinflammatory pathology presents as interferonopathy, but in mice is interferon-independent.

BACKGROUND: Autoimmune diseases are leading causes of ill health and morbidity and have diverse etiology. Two signaling pathways are key drivers of autoimmune pathology, interferon and nuclear factor-κB (NF-κB)/RelA, defining the 2 broad labels of interferonopathies and relopathies. Prior work has established that genetic loss of function of the NF-κB subunit RelB leads to autoimmune and inflammatory pathology in mice and humans. OBJECTIVE: We sought to characterize RelB-deficient autoimmunity by unbiased profiling of the responses of immune sentinel cells to stimulus and to determine the functional role of dysregulated gene programs in the RelB-deficient pathology. METHODS: Transcriptomic profiling was performed on fibroblasts and dendritic cells derived from patients with RelB deficiency and knockout mice, and transcriptomic responses and pathology were assessed in mice deficient in both RelB and the type I interferon receptor. RESULTS: We found that loss of RelB in patient-derived fibroblasts and mouse myeloid cells results in elevated induction of hundreds of interferon-stimulated genes. Removing hyperexpression of the interferon-stimulated gene program did not ameliorate the autoimmune pathology of RelB knockout mice. Instead, we found that RelB suppresses a different set of inflammatory response genes in a manner that is independent of interferon signaling but associated with NF-κB binding motifs. CONCLUSION: Although transcriptomic profiling would describe RelB-deficient autoimmune disease as an interferonopathy, the genetic evidence indicates that the pathology in mice is interferon-independent.

NFκB pathway dysregulation due to reduced RelB expression leads to severe autoimmune disorders and declining immunity.

BACKGROUND: Genetic aberrations in the NFκB pathway lead to primary immunodeficiencies with various degrees of severity. We previously demonstrated that complete ablation of the RelB transcription factor, a key component of the alternative pathway, results in an early manifested combined immunodeficiency requiring stem cell transplantation. OBJECTIVE: To study the molecular basis of a progressive severe autoimmunity and immunodeficiency in three patients. METHODS: Whole exome sequencing was performed to identify the genetic defect. Molecular and cellular techniques were utilized to assess the variant impact on NFκB signaling, canonical and alternative pathway crosstalk, as well as the resultant effects on immune function. RESULTS: Patients presented with multiple autoimmune progressive severe manifestations encompassing the liver, gut, lung, and skin, becoming debilitating in the second decade of life. This was accompanied by a deterioration of the immune system, demonstrating an age-related decline in naïve T cells and responses to mitogens, accompanied by a gradual loss of all circulating CD19+ cells. Whole exome sequencing identified a novel homozygous c. C1091T (P364L) transition in RELB. The P364L RelB protein was unstable, with extremely low expression, but retained some function and could be transiently and partially upregulated following Toll-like receptor stimulation. Stimulation of P364L patient fibroblasts resulted in a marked rise in a cluster of pro-inflammatory hyper-expressed transcripts consistent with the removal of RelB inhibitory effect on RelA function. This is likely the main driver of autoimmune manifestations in these patients. CONCLUSION: Incomplete loss of RelB provided a unique opportunity to gain insights into NFκB's pathway interactions as well as the pathogenesis of autoimmunity. The P364L RelB mutation leads to gradual decline in immune function with progression of severe debilitating autoimmunity.

BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown. METHODS: BCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24-, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment. RESULTS: BRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24- and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24- and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells. CONCLUSIONS: The results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC. Video Abstract.

Aryl hydrocarbon receptor (AhR) reveals evidence of antagonistic pleiotropy in the regulation of the aging process.

The antagonistic pleiotropy hypothesis is a well-known evolutionary theory to explain the aging process. It proposes that while a particular gene may possess beneficial effects during development, it can exert deleterious properties in the aging process. The aryl hydrocarbon receptor (AhR) has a significant role during embryogenesis, but later in life, it promotes several age-related degenerative processes. For instance, AhR factor (i) controls the pluripotency of stem cells and the stemness of cancer stem cells, (ii) it enhances the differentiation of embryonal stem cells, especially AhR signaling modulates the differentiation of hematopoietic stem cells and progenitor cells, (iii) it also stimulates the differentiation of immunosuppressive Tregs, Bregs, and M2 macrophages, and finally, (iv) AhR signaling participates in the differentiation of many peripheral tissues. On the other hand, AhR signaling is involved in many processes promoting cellular senescence and pathological processes, e.g., osteoporosis, vascular dysfunction, and the age-related remodeling of the immune system. Moreover, it inhibits autophagy and aggravates extracellular matrix degeneration. AhR signaling also stimulates oxidative stress, promotes excessive sphingolipid synthesis, and disturbs energy metabolism by catabolizing NAD+ degradation. The antagonistic pleiotropy of AhR signaling is based on the complex and diverse connections with major signaling pathways in a context-dependent manner. The major regulatory steps include, (i) a specific ligand-dependent activation, (ii) modulation of both genetic and non-genetic responses, (iii) a competition and crosstalk with several transcription factors, such as ARNT, HIF-1α, E2F1, and NF-κB, and (iv) the epigenetic regulation of target genes with binding partners. Thus, not only mTOR signaling but also the AhR factor demonstrates antagonistic pleiotropy in the regulation of the aging process.

A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection.

A hallmark of infection by the pathogen Helicobacter pylori, which colonizes the human gastric epithelium, is the simultaneous activation of the classical and alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, underlying inflammation and cell survival. Here, we report that the classical NF-κB target gene product A20 contributes to the negative regulation of alternative NF-κB signaling in gastric epithelial cells infected by H. pylori. Mechanistically, the de novo synthesized A20 protein interacts with tumor necrosis factor receptor-associated factor-interacting protein with forkhead-associated domain (TIFA) and thereby interferes with the association of TIFA with the NIK regulatory complex. We also show that alternative NF-κB activity contributes to the up-regulation of anti-apoptotic genes, such as baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1) in gastric epithelial cells. Furthermore, the observed over-expression of RelB in human gastric biopsies with type B gastritis and RelB-dependent suppression of apoptotic cell death emphasize an important role of the alternative NF-κB pathway in H. pylori infection.

Inflammation in Hypervolemic Hemodialysis Patients: The Roles of RelB and Caspase-4.

Hypervolemia is associated with inflammation in hemodialysis (HD) patients. How hypervolemia triggers inflammation is not entirely known. We initiated a cross-sectional study enrolling 40 hemodialysis patients who were categorized into normovolemic (N; 23) and hypervolemic (H; 17) groups by bioimpedance measurement. A caspase activity assay in combination with a specific caspase-4 inhibitor was used to detect caspase-4 activity in isolated peripheral blood mononuclear cells (PBMCs). Transcription factors RelA (pS529) and RelB (pS552) were analyzed by phospho-flow cytometry. Serum endotoxins were detected by an amebocyte lysate-based assay, and IL-6 (interleukin-6) and TNF-α (Tumor necrosis factor-α) gene expression were detected using the ELISA technique. Hypervolemic patients were older, more frequently had diabetes and showed increased CRP and IL-6 levels. Caspase-4 activity, which is linked to intracellular endotoxin detection, was significantly elevated in H patients. While the frequency of RelA-expressing immune cells and the expression density in these cells did not differ, the monocytic frequency of cells positively stained for RelB (pS552) was significantly decreased in H patients. Increased caspase-4 activity in H patients may indicate a cause of inflammation in H patients. The post-translational modification of RelB (pS552) is linked to downregulation of NF-kB activity and may indicate the resolution of inflammation, which is more distinct in N patients compared to H patients. Therefore, both higher inflammatory loads and lower inflammatory resolution capacities are characteristics of H patients.

NF-κB Signaling Modulates miR-452-5p and miR-335-5p Expression to Functionally Decrease Epithelial Ovarian Cancer Progression in Tumor-Initiating Cells.

Epithelial ovarian cancer (EOC) remains the fifth leading cause of cancer-related death in women worldwide, partly due to the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. We previously described a role for the NF-κB pathway in promoting TIC chemoresistance and survival through NF-κB transcription factors (TFs) RelA and RelB, which regulate genes important for the inflammatory response and those associated with cancer, including microRNAs (miRNAs). We hypothesized that NF-κB signaling differentially regulates miRNA expression through RelA and RelB to support TIC persistence. Inducible shRNA was stably expressed in OV90 cells to knockdown RELA or RELB; miR-seq analyses identified differentially expressed miRNAs hsa-miR-452-5p and hsa-miR-335-5p in cells grown in TIC versus adherent conditions. We validated the miR-seq findings via qPCR in TIC or adherent conditions with RELA or RELB knocked-down. We confirmed decreased expression of hsa-miR-452-5p when either RELA or RELB were depleted and increased expression of hsa-miR-335-5p when RELA was depleted. Either inhibiting miR-452-5p or mimicking miR-335-5p functionally decreased the stem-like potential of the TICs. These results highlight a novel role of NF-κB TFs in modulating miRNA expression in EOC cells, thus opening a better understanding toward preventing recurrence of EOC.

Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa.

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

The RelB-BLNK Axis Determines Cellular Response to a Novel Redox-Active Agent Betamethasone during Radiation Therapy in Prostate Cancer.

Aberrant levels of reactive oxygen species (ROS) are potential mechanisms that contribute to both cancer therapy efficacy and the side effects of cancer treatment. Upregulation of the non-canonical redox-sensitive NF-kB family member, RelB, confers radioresistance in prostate cancer (PCa). We screened FDA-approved compounds and identified betamethasone (BET) as a drug that increases hydrogen peroxide levels in vitro and protects non-PCa tissues/cells while also enhancing radiation killing of PCa tissues/cells, both in vitro and in vivo. Significantly, BET increases ROS levels and exerts different effects on RelB expression in normal cells and PCa cells. BET induces protein expression of RelB and RelB target genes, including the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), in normal cells, while it suppresses protein expression of RelB and MnSOD in LNCaP cells and PC3 cells. RNA sequencing analysis identifies B-cell linker protein (BLNK) as a novel RelB complementary partner that BET differentially regulates in normal cells and PCa cells. RelB and BLNK are upregulated and correlate with the aggressiveness of PCa in human samples. The RelB-BLNK axis translocates to the nuclear compartment to activate MnSOD protein expression. BET promotes the RelB-BLNK axis in normal cells but suppresses the RelB-BLNK axis in PCa cells. Targeted disruptions of RelB-BLNK expressions mitigate the radioprotective effect of BET on normal cells and the radiosensitizing effect of BET on PCa cells. Our study identified a novel RelB complementary partner and reveals a complex redox-mediated mechanism showing that the RelB-BLNK axis, at least in part, triggers differential responses to the redox-active agent BET by stimulating adaptive responses in normal cells but pushing PCa cells into oxidative stress overload.

Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation.

Ectodysplasin A (Eda) signaling activates NF-κB during skin appendage formation, but how Eda controls specific gene transcription remains unclear. Here, we find that Eda triggers the formation of an NF-κB-associated SWI/SNF (BAF) complex in which p50/RelB recruits a linker protein, Tfg, that interacts with BAF45d in the BAF complex. We further reveal that Tfg is initially induced by Eda-mediated RelB activation and then bridges RelB and BAF for subsequent gene regulation. The BAF component BAF250a is particularly up-regulated in skin appendages, and epidermal knockout of BAF250a impairs skin appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several target genes regulated by Eda, RelB, and BAF. Notably, RelB and the BAF complex are indispensable for transcription of Eda target genes, and both BAF complex and Eda signaling are required to open chromatin of Eda targets. Our studies thus suggest that Eda initiates a signaling cascade and recruits a BAF complex to specific gene loci to facilitate transcription during organogenesis.

Identification of undescribed Relb expression domains in the murine brain by new Relb:cre-katushka reporter mice.

BACKGROUND: Noncanonical NF-κB signaling through activation of the transcription factor RelB acts as key regulator of cell lineage determination and differentiation in various tissues including the immune system. To elucidate temporospatial aspects of Relb expression, we generated a BAC transgenic knock-in mouse expressing the fluorescent protein Katushka and the enzyme Cre recombinase under control of the murine Relb promoter (RelbCre-Kat mice). RESULTS: Co-expression of Katushka and Relb in fibroblast cultures and tissues of transgenic mice revealed highly specific reporter functions of the transgene. Crossing RelbCre-Kat mice with ROSA26R reporter mice that allow for Cre-mediated consecutive ß-galactosidase or YFP synthesis identified various Relb expression domains in perinatal and mature mice. Besides thymus and spleen, highly specific expression patterns were found in different neuronal domains, as well as in other nonimmune organs including skin, skeletal structures and kidney. De novo Relb expression in the mature brain was confirmed in conditional knockout mice with neuro-ectodermal Relb deletion. CONCLUSION: Our results demonstrate the usability of RelbCre-Kat reporter mice for the detection of de novo and temporarily restricted Relb expression including cell and lineage tracing of Relb expressing cells. Relb expression during mouse embryogenesis and at adulthood suggests, beyond immunity, important functions of this transcription factor in neurodevelopment and CNS function.

RelB promotes liver fibrosis via inducing the release of injury-associated inflammatory cytokines.

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-ß1 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.

RelB regulates the homeostatic proliferation but not the function of Tregs.

BACKGROUND: RelB, a member of the NF-κB family, plays a critical role in the development of T cells. However, the role of RelB in Foxp3+ regulatory T cells (Tregs) remains controversial. RESULTS: Using a bone marrow chimeric mouse model, we demonstrated that the expansion of Foxp3+ Tregs in vivo could be mediated by extrinsic mechanisms. RelB plays an important role in inhibiting the homeostatic proliferation of Tregs, but not their survival. Even with the heightened expansion, RelB-/- Treg cells displayed normal suppressive function in vitro. Among the expanded populations of Treg cells, most were nTreg cells; however, the population of iTregs did not increase. Mechanistically, RelB seems to regulate Treg proliferation independently of the signal transducer and activator of transcription 5 (STAT5) pathway. CONCLUSIONS: These data suggest that RelB regulates Treg proliferation independently of the STAT5 pathway, but does not alter the function of Tregs. Further studies are warranted to uncover such mechanisms.

RelB suppresses type I Interferon signaling in dendritic cells.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

CEACAMs interaction with Helicobacter pylori HopQ supports the type 4 secretion system-dependent activation of non-canonical NF-κB.

Helicobacter pylori infection represents a major risk factor for the development of gastric diseases and gastric cancer. The capability of H. pylori to inject the virulence factor cytotoxin-associated gene A (CagA) depends on a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). Further, infection by H. pylori activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in a T4SS-dependent manner but CagA-independent manner. Here we investigated the role of host cell receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the bacterial adhesin HopQ in the activation of non-canonical NF-κB and CagA translocation into gastric epithelial cells. AGS cells express six of twelve CEACAMs found in humans. In HeLa cells, only CEACAM19 is expressed. We showed that deletion of hopQ attenuates the activation of non-canonical NF-κB only in AGS but not in HeLa cells. CagA translocation was in both cell lines affected by HopQ depletion, although to a much lesser extent in HeLa cells. Moreover, we observed a possible redundancy between the three HopQ-binding CEACAMs 1, 5 and 6 and their capacity to support non-canonical NF-κB activation. Our results illustrate that the interaction between HopQ and CEACAMs could promote the efficiency of the T4SS.

RelB sustains endocrine resistant malignancy: an insight of noncanonical NF-κB pathway into breast Cancer progression.

BACKGROUND: The activation of the NF-κB pathway plays a crucial role in the progression of breast cancer (BCa) and also involved in endocrine therapy resistance. On the contrary to the canonical NF-κB pathway, the effect of the noncanonical NF-κB pathway in BCa progression remains elusive. METHODS: BCa tumor tissues and the corresponding cell lines were examined to determine the correlation between RelB and the aggressiveness of BCa. RelB was manipulated in BCa cells to examine whether RelB promotes cell proliferation and motility by quantitation of apoptosis, cell cycle, migration, and invasion. RNA-Seq was performed to identify the critical RelB-regulated genes involved in BCa metastasis. Particularly, RelB-regulated MMP1 transcription was verified using luciferase reporter and ChIP assay. Subsequently, the effect of RelB on BCa progression was further validated using BCa mice xenograft models. RESULTS: RelB uniquely expresses at a high level in aggressive BCa tissues, particularly in triple-negative breast cancer (TNBC). RelB promotes BCa cell proliferation through increasing G1/S transition and/or decreasing apoptosis by upregulation of Cyclin D1 and Bcl-2. Additionally, RelB enhances cell mobility by activating EMT. Importantly, RelB upregulates bone metastatic protein MMP1 expression through binding to an NF-κB enhancer element located at the 5'-flanking region. Accordingly, in vivo functional validation confirmed that RelB deficiency impairs tumor growth in nude mice and inhibits lung metastasis in SCID mice. Video abstract.

RelB controls adaptive responses of astrocytes during sterile inflammation.

In response to brain injury or infections, astrocytes become reactive, undergo striking morphological and functional changes, and secrete and respond to a spectrum of inflammatory mediators. We asked whether reactive astrocytes also display adaptive responses during sterile IL-1ß-induced neuroinflammation, which may limit tissue injury associated with many disorders of the central nervous system. We found that astrocytes display days-to-weeks long specific tolerance of cytokine genes, which is coordinated by NF-κB family member, RelB. However, in contrast to innate immune cells, astrocytic tolerance does not involve epigenetic silencing of the cytokine genes. Establishment of tolerance depends on persistent higher levels of RelB in tolerant astrocytes and its phosphorylation on serine 472. Mechanistically, this phosphorylation prevents efficient removal of RelB from cytokine promoters by IκBα and helps to establish tolerance. Importantly, ablation of RelB from astrocytes in mice abolishes tolerance during experimental neuroinflammation in vivo.