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Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome results from NFKB2 heterozygous mutations, causing adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia. While NFKB signaling plays a crucial role in the immune system, its connection to endocrine symptoms is unclear. We established a human disease model to investigate the role of NFKB2 in pituitary development by creating pituitary organoids from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSCs). Introducing homozygous TBX19 K146R/K146R missense pathogenic variant in hiPSC, an allele found in congenital isolated ACTHD, led to a strong reduction of corticotrophs number in pituitary organoids. Then, we characterized the development of organoids harboring NFKB2 D865G/D865G mutations found in DAVID patients. NFKB2 D865G/D865G mutation acted at different levels of development with mutant organoids displaying changes in the expression of genes involved on pituitary progenitor generation ( HESX1 , PITX1 , LHX3 ), hypothalamic secreted factors ( BMP4, FGF8, FGF10 ), epithelial-to-mesenchymal transition, lineage precursors development ( TBX19 , POU1F1 ) and corticotrophs terminal differentiation ( PCSK1, POMC ), and showed drastic reduction in the number of corticotrophs. Our results provide strong evidence for the direct role of NFKB2 mutations in the endocrine phenotype observed in patients leading to a new classification of a NFKB2 variant of previously unknown clinical significance as pathogenic in pituitary development., Competing Interests: TM, TF, NJ, PR, HE, AB, FC, TB No competing interests declared, (© 2023, Mac et al.)

Purpose: High-pressure-processed (HPP) rice is white rice that maintains some key functional food ingredients of brown rice, such as polyphenols. We examined whether HPP rice intake during interval walking training (IWT) improved glycemic control with enhanced methylation of the NFKB2 gene in hyperglycemic older subjects., Methods: We recruited 51 people aged ~ 70 yr who had already performed IWT for ≥ 6 months, but had hyperglycemia (blood glucose concentration ([Glc]) > 110 mg/dl or HbA1c > 6.0% while fasting). Participants were randomly divided into control (CNT) or HPP rice (HPR) groups and instructed to perform IWT for an additional 4 months while ingesting 75 g dry weight of either white rice or HPP rice, respectively, at every breakfast and dinner. Before and after intervention, [Glc] was measured by continuous glucose monitoring for 5 days, with standardized breakfast on day 5. Methylation of NFKB2 was measured by pyrosequencing., Results: After intervention, mean fasting [Glc] values for 180 min before breakfast over 4 days (days 2-5) marginally decreased in HPR but were not different from CNT (P = 0.17). However, the standard deviation during the period decreased more in HPR than in CNT (P = 0.013). Moreover, total area under the curve (tAUC) for 180 min after breakfast on day 5 decreased more in HPR than in CNT (P = 0.035). The change in tAUC on day 5 after the intervention was negatively correlated with that in NFKB2 gene methylation (P = 0.002)., Conclusion: HPP rice intake during IWT improved glycemic control with suppressed reduction in NFKB2 gene methylation in hyperglycemic older people., Trial Registration Number and Date of Registration: UMIN000024390; October 13, 2016., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest. Ethical approval: This study was approved by the Review Board on Human Experiments, Shinshu University School of Medicine (approval no. 3542), and it conformed to the standards set by the Declaration of Helsinki. The protocol was registered with the University Hospital Medical Information Network (UMIN) in Japan, (trial registration number: UMIN000024390) on October 13, 2016., (© 2024. The Author(s).)

The complex heterogeneity of tumor microenvironment (TME) of triple-negative breast cancer (TNBC) presents a significant obstacle to cytotoxic immune response and successful treatment, building up one of the most hostile oncological phenotypes. Among the most abundant TME components, tumor-associated macrophages (TAMs) have pivotal pro-tumoral functions, involving discordant roles for the nuclear factor kappa-B (NF-κB) transcription factors and directing to higher levels of pathway complexity. In both resting macrophages and TAMs, we recently revealed the existence of the uncharacterized NF-κB p65/p52 dimer. In the present study, we demonstrated its enhanced active nuclear localization in TAMs and validated selected immune target genes as directly regulated by dimer binding on DNA sequences. We demonstrated by ChIP-qPCR that p65/p52 enrichment on HSPG2 and CSF-1 regulatory regions is strictly dependent on macrophage polarization and tumor environment. Our data provide novel mechanisms of transcriptional regulation in TAMs, orchestrated by the varied and dynamic nature of NF-κB combinations, which needs to be considered when targeting this pathway in cancer therapies. Our results offer p65/p52, together with identified regulatory regions on genes impacting macrophage behavior and tumor biology, as novel molecular targets for TNBC, aimed at modulating TAMs functions towards anti-tumoral phenotypes and thus improving cancer treatment outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The NF-κB family consists of the key transcription factors of the NF-κB signaling pathway, well known for its role in innate immune response, organogenesis, and a variety of cellular processes. The five NF-κB subunits-RelA, RelB, c-Rel, p50, and p52-are functional dimers, each of which share a conserved DNA binding domain which contains the dimerization domain (DD) as well. The NF-κB subunits can form 15 potential dimers among themselves of which, RelA-p50 is extensively studied and has largely become synonymous with NF-κB for transcription activation. While various reports have highlighted the importance of NF-κB subunit specificity in the transcription regulation of certain target genes, the dynamic nature of the NF-κB dimer composition is not well understood. In this study, we biophysically characterized six combinatorial dimers from three NF-κB subunits: RelA, p50, and p52, using NMR spectroscopy and differential scanning calorimetry. We show that the dimer composition is dynamic and can readily undergo exchange although at varied rates. Among the six dimers formed, RelA-p52 is found to be the most stable dimer with RelA-RelA being the least. Our results provide a plausible explanation as to why the RelA-p52 heterodimer is active during the later stages of the NF-κB activation and serve as a link between the canonical and non-canonical NF-κB pathway., (© 2024 The Protein Society.)

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss-of-function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B ( RELB). The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-κB2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild-type RELB complementary DNA (cDNA). By contrast, the response of RelB-deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL-1β via the canonical NF-κB pathway remains intact. P1 and P2 have low proportions of naïve CD4 + and CD8 + T cells and of memory B cells. Moreover, their naïve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)-secreting cells in response to CD40L/IL-21, and the development of IL-17A/F-producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-κB pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections., Competing Interests: Competing interests statement:Tim Niehues was a co-author on publications with the following co-authors within the last 4 y: K.B., C.S.M., and S.G.T. (https://doi.org/10.1182/blood.2020006738) and K.B. (https://doi.org/10.1016/j.jaci.2019.11.051; https://doi.org/10.1002/eji.202048713). Andrew R. Gennery was a co-author with S.G.T. (https://doi.org/10.1016/j.jaci.2020.09.010; https://doi.org/10.1016/j.jaci.2022.09.002) within the last 4 y.

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 β-catenin destruction complex, reinforcing β-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 β-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., (© 2024. The Author(s).)

Objectives: The mechanism that leads to disseminated tuberculosis in HIV-negative patients is still largely unknown. T cell subsets and signaling pathways that were associated with disseminated tuberculosis were investigated., Methods: Single-cell profiling of whole T cells was performed to identify T cell subsets and enriched signaling pathways that were associated with disseminated tuberculosis. Flow cytometric analysis and blocking experiment were used to investigate the findings obtained by transcriptome sequencing., Results: Patients with disseminated tuberculosis had depleted Th1, Tc1 and Tc17 cell subsets, and IFNG was the most down-regulated gene in both CD4 and CD8 T cells. Gene Ontology analysis showed that non-canonical NF-κB signaling pathway, including NFKB2 and RELB genes, was significantly down-regulated and was probably associated with disseminated tuberculosis. Expression of several TNF superfamily ligands and receptors, such as LTA and TNF genes, were suppressed in patients with disseminated tuberculosis. Blocking of TNF-α and soluble LTα showed that TNF-α was involved in IFN-γ production and LTα influenced TNF-α expression in T cells., Conclusions: Impaired T cell IFN-γ response mediated by suppression of TNF and non-canonical NF-κB signaling pathways might be responsible for disseminated tuberculosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nuclear factor kappa B (NF-κB) family plays a central role in the human immune system. Heterozygous variants in NFKB2 typically cause immunodeficiency with various degrees of central adrenal insufficiency, autoimmunity, and ectodermal dysplasia. No reported case has presented kidney failure as an initial symptom. Moreover, documentation of kidney involvement of this disease is limited. CASE DIAGNOSIS: A 2-year-old female who presented with dyspnea and hypertensive emergency in the setting of new-onset nephrotic syndrome with acute-on chronic kidney injury with resultant chronic kidney disease (CKD) was found to have a novel heterozygous N-terminal variant in NFKB2 (c.880del: p. Tyr294Ilefs*4) with mild hypogammaglobulinemia, but no adrenal insufficiency or ectodermal dysplasia. She became dialysis-dependent during her initial hospitalization and developed CKD stage 5D, requiring continued peritoneal dialysis. She is currently awaiting kidney transplantation. CONCLUSIONS: Whether nephrotic syndrome or kidney injury or failure is the primary symptom of this variant or secondary to some event remains unknown. Further case accumulation is warranted., (© 2024. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Background: Inborn errors of immunity (IEIs) encompass various diseases with diverse clinical and immunological symptoms. Determining the genotype-phenotype of different variants in IEI entity precisely is challenging, as manifestations can be heterogeneous even in patients with the same mutated gene., Objective: In the present study, we conducted a systematic review of patients recorded with NFKB1 and NFKB2 mutations, two of the most frequent monogenic IEIs., Methods: The search for relevant literature was conducted in databases including Web of Science, PubMed, and Scopus. Information encompassing demographic, clinical, immunological, and genetic data was extracted from cases reported with mutations in NFKB1 and NFKB2. The comprehensive features of manifestations in patients were described, and a comparative analysis of primary characteristics was conducted between individuals with NFKB1 loss of function (LOF) and NFKB2 (p52-LOF/IκBδ-gain of function (GOF)) variants., Results: A total of 397 patients were included in this study, 257 had NFKB1 mutations and 140 had NFKB2 mutations. There were 175 LOF cases in NFKB1 and 122 p52 LOF /IκBδ GOF cases in NFKB2 pivotal groups with confirmed functional implications. NFKB1LOF and p52 LOF /IκBδ GOF predominant cases (81.8% and 62.5% respectively) initially presented with a CVID-like phenotype. Patients with NFKB1LOF variants often experienced hematologic autoimmune disorders, whereas p52 LOF /IκBδ GOF patients were more susceptible to other autoimmune diseases. Viral infections were markedly higher in p52 LOF /IκBδ GOF cases compared to NFKB1LOF (P-value < 0.001). NFKB2 (p52 LOF /IκBδ GOF ) patients exhibited a greater prevalence of ectodermal dysplasia and pituitary gland involvement than NFKB1LOF patients. Most NFKB1LOF and p52 LOF /IκBδ GOF cases showed low CD19 + B cells, with p52 LOF /IκBδ GOF having more cases of this type. Low memory B cells were more common in p52 LOF /IκBδ GOF patients., Conclusions: Patients with NFKB2 mutations, particularly p52 LOF /IκBδ GOF , are at higher risk of viral infections, pituitary gland involvement, and ectodermal dysplasia compared to patients with NFKB1LOF mutations. Genetic testing is essential to resolve the initial complexity and confusion surrounding clinical and immunological features. Emphasizing the significance of functional assays in determining the probability of correlations between mutations and immunological and clinical characteristics of patients is crucial., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs) 1,2 , conferring a predisposition to life-threatening COVID-19 pneumonia 3 . Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52 LOF /IκBδ GOF ). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52 LOF /IκBδ LOF ) or gain-of-function of p52 (hereafter, p52 GOF /IκBδ LOF ). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases., (© 2023. The Author(s).)

The ubiquitination-dependent processing of NF-κB2 (also known as p100) is a critical step in the activation of the noncanonical NF-κB pathway. We investigated the molecular mechanisms regulating this process and showed that TRIM55 was the E3 ubiquitin ligase that mediated the ubiquitination of p100 and coordinated its processing. TRIM55 deficiency impaired noncanonical NF-κB activation and B cell function. Mice with a B cell-specific Trim55 deficiency exhibited reduced germinal center formation and antibody production. These mice showed less severe symptoms than those of control mice upon the induction of a systemic lupus-like disease, suggesting B cell-intrinsic functions of TRIM55 in humoral immune responses and autoimmunity. Mechanistically, the ubiquitination of p100 mediated by TRIM55 was crucial for p100 processing by VCP, an ATPase that mediates ubiquitin-dependent protein degradation by the proteasome. Furthermore, we found that TRIM55 facilitated the interaction between TRIM21 and VCP as well as TRIM21-mediated K63-ubiquitination of VCP, both of which were indispensable for the formation of the VCP-UFD1-NPL4 complex and p100 processing. Together, our results reveal a mechanism by which TRIM55 fine-tunes p100 processing and regulates B cell-dependent immune responses in vivo, highlighting TRIM55 as a potential therapeutic target for lupus-like disease.

Introduction: Genetic defects affect the manner of the immune system's development, activation, and function. Nuclear factor-kappa B subunit 1 (NF-κB1) and NF-κB2 are involved in different biological processes, and deficiency in these transcription factors may reveal clinical and immunological difficulties., Areas Covered: This review article gathers the most frequent clinical and immunological remarkable characteristics of NF-κB1 and NF-κB2 deficiencies. Afterward, an effort is made to describe the biological mechanism, which is likely to be the cause of these clinical and immunological abnormalities., Expert Opinion: The present review article has explained the mechanism of contributions of the NF-κB1 and NF-κB2 deficiency in revealing immunodeficiency symptoms, specifically immunological and clinical manifestations. These mechanisms demonstrate the importance of NF-κB1 and NF-κB2 signaling pathways for B and T cell development, activation, antibody production, and immunotolerance. The manifestation of a mutation can range from no symptoms to severe complications in a family.

Objectives: Sudden Sensorineural Hearing Loss (SSNHL) is an increasingly common health problem today. Although the direct mortality rate of this disorder is relatively low, its impact on quality of life is enormous; this is why accurate identification of pathogenesis and influencing factors in the disease process can play an essential role in preventing and treating the disease. Acute inflammation, which leads to chronic inflammation due to aberrant expression of inflammation-mediating genes, may play a significant role in the pathogenesis of the disease. The essential Nuclear factor kappa B (NF-kB) pathway genes, NFKB1 and NFKB2, serve as prothrombotic agents when expressed abnormally, compromising the cochlea by disrupting the endolymphatic potential and causing SSNHL., Methods: This study investigates the expression levels of NFKB1 and NFKB2 in peripheral blood (PB) through a quantitative polymerase chain reaction in 50 Iranian patients with SSNHL, and 50 healthy volunteers were of the same age and sex as controls., Results: As a result, NFKB2 expression levels in patients were higher than in controls, regardless of sex or age (posterior beta = 0.619, adjusted P-value = 0.016), and NFKB1 expression levels did not show significant differences between patients and controls. The expression levels of NFKB1 and NFKB2 had significantly strong positive correlations in both SSNHL patients and healthy individuals (r = 0.620, P = 0.001 and r = 0.657, P 0.001, respectively), suggesting the presence of an interconnected network., Conclusion: NFKB2 has been identified as a significant inflammatory factor in the pathophysiology of SSNHL disease. Inflammation can play an essential role in developing SSNHL, and our findings could be used as a guide for future research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb 2 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.

Background: Patients with relapsed/refractory acute myeloid leukaemia (AML) with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) have limited treatment options and poor prognosis. Therefore, novel treatment modalities are needed. Since high expression of natural killer group 2 member D ligands (NKG2DLs) can be induced by FLT3 inhibitors, we constructed dual-target FLT3 single-chain fragment variable (scFv)/NKG2D-chimeric antigen receptor (CAR) T cells, and explored whether FLT3 inhibitors combined with FLT3scFv/NKG2D-CAR T cells could have synergistic anti-leukaemia effects., Methods: FLT3scFv and NKG2D expression in CAR T cells, FLT3 and NKG2DL expression in AML cells, and the in vitro cytotoxicity of combining CAR T cells with gilteritinib were assessed by flow cytometry. The therapeutic effect was evaluated in a xenograft mouse model established by injection of MOLM-13 cells. Mechanisms underlying the gilteritinib-induced NKG2DL upregulation were investigated using siRNA, ChIP-QPCR and luciferase assays., Results: The FLT3scFv/NKG2D-CAR T cells specifically lysed AML cells both in vitro and in the xenograft mouse model. The efficacy of FLT3scFv/NKG2D-CAR T cells was improved by gilteritinib-pretreatment. The noncanonical NF-κB2/Rel B signalling pathway was found to mediate gilteritinib-induced NKG2DL upregulation in AML cells., Conclusions: Bispecific FLT3scFv/NKG2D-CAR T cells can effectively eradicate AML cells. The FLT3 inhibitor gilteritinib can synergistically improve this effect by upregulating NF-κB2-dependent NKG2DL expression in AML cells., (© 2022. The Author(s).)

Introduction: Although most cases of coccidioidomycosis are subclinical or self-limited respiratory disease, 1% lead to extrathoracic dissemination and become fatal, especially in patients with an associated immunodeficiency. Up to 30%-50% of patients with defects in cell-mediated immunity, those with AIDS and recipients of solid-organ transplants, may develop disseminated coccidioidomycosis (DC). Within the primary immunodeficiencies, an uncommon group is caused by C-terminal NFKB2 pathogenic variants., Materials and Methods: We performed a literature search of core databases. Written informed consent for the study and for publication was obtained., Case Presentation: A 7-year-old Mexican girl, eldest of 3 sisters, no relevant family history, and a history of recurrent upper respiratory infections and alopecia totalis was admitted with DC involving pulmonary, soft tissue, skin, bone and joint compromise. The immunodeficiency assessment showed low IgM and NK cells. We found an NFKB2 de novo heterozygous nonsense mutation of c.2611C>T (p.Gln871*). She was treated with liposomal amphotericin B and itraconazole with surgical debridement. The clinical phenotype of this primary immunodeficiency is characterized by antibody deficiency and associated broncho-pulmonary predisposition to infection, but moreover also opportunistic infections and autoimmunity, most recognizable alopecia and adrenocorticotropic hormone-deficiency. After 1 year of her discharge, she continues under surveillance with antifungal therapy with itraconazole and replacement intravenous immunoglobulin until today., Conclusion: This is the first case report of DC in a patient with an NFKB2 pathogenic variant and it illustrates the importance of screening for primary immunodeficiencies in patients with disseminated fungal infections., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Extracellular matrix degradation, reactive oxygen species (ROS) generation, and inflammation in nucleus pulposus (NP) cells contribute to the progression of intervertebral disc degeneration (IDD). NRGs (Neuregulins) play a vital role in the development of the nervous system. In the present study, we found that NRG1 was downregulated within degenerative intervertebral disc and NP tissues, according to both bioinformatics and experimental analyses. Within IL-1β-stimulated NP cells, we observed degenerative and inflammatory changes, including inhibited cell viability, promoted cell apoptosis and ROS accumulation, reduced collagen II and aggrecan proteins, elevated MMP-3/13 and ADAMTS-4/5 proteins, and upregulated IL-6 and TNF-α mRNA levels. Within IL-1β-stimulated NP cells, NRG1 expression was also downregulated. NRG1 overexpression attenuated, whereas NRG1 silencing aggravated IL-1β-induced degenerative and inflammatory changes. Moreover, NRG1 regulated ErbB2/3 activation, contributing to the NRG1 protective function in NP cells. NFKB2 directly targeted the promoter region of NRG1 and inhibited NRG1 expression. In IL-1β-stimulated NP cells, silencing NFKB2 attenuated, whereas silencing NRG1 aggravated the degenerative changes and inflammation; the effects of NFKB2 silencing were significantly reversed by NRG1 silencing. In conclusion, NRG1 expression is downregulated within degenerative NP tissue samples and IL-1β-stimulated NP cells. NRG1 might protect against IL-1β-induced degenerative changes and inflammation. The upregulated NFKB2 might be the reason of NRG1 downregulation in degenerative NP tissues., (Copyright © 2021 Elsevier B.V. All rights reserved.)

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100., Competing Interests: Disclosures: M.D. Keller reported "other" from Gilead outside the submitted work. D.H.D. Gray reported grants from Servier Pharmacetuicals outside the submitted work and is an employee of The Walter and Eliza Hall Institute of Medical Research, which receives milestone and royalty payments related to venetoclax (BCL-2 inhibitor). No other disclosures were reported., (© 2020 Wirasinha et al.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged recently and has created a global pandemic. Symptomatic SARS-CoV-2 infection, termed coronavirus disease 2019 (COVID-19), has been associated with a host of symptoms affecting numerous organ systems, including the lungs, cardiovascular system, kidney, central nervous system, gastrointestinal tract, and skin, among others., Objective: Although several risk factors have been identified as related to complications from and severity of COVID-19, much about the virus remains unknown. The host immune response appears to affect the outcome of disease. It is not surprising that patients with intrinsic or secondary immune compromise might be particularly susceptible to complications from SARS-CoV-2 infection. Pathogenic loss-of-function or gain-of-function heterozygous variants in nuclear factor-κB2 have been reported to be associated with either a combined immunodeficiency or common variable immunodeficiency phenotype., Methods: We evaluated the functional consequence and immunologic phenotype of a novel NFKB2 loss of function variant in a 17-year-old male patient and describe the clinical management of SARS-CoV-2 infection in this context., Results: This patient required a 2-week hospitalization for SARS-CoV-2 infection, including 7 days of mechanical ventilation. We used biologic therapies to avert potentially fatal acute respiratory distress syndrome and treat hyperinflammatory responses. The patient had an immunologic phenotype of B-cell dysregulation with decreased switched memory B cells. Despite the underlying immune dysfunction, he recovered from the infection with intense management., Conclusions: This clinical case exemplifies some of the practical challenges in management of patients with SARS-CoV-2 infection, especially in the context of underlying immune dysregulation., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G > A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

Background: Bromodomain and extra-terminal domain (BET) inhibitors have shown profound efficacy against hematologic malignancies and solid tumors in preclinical studies. However, the underlying molecular mechanism in melanoma is not well understood. Here we identified secreted phosphoprotein 1 (SPP1) as a melanoma driver and a crucial target of BET inhibitors in melanoma. Methods: Bioinformatics analysis and meta-analysis were used to evaluate the SPP1 expression in normal tissues, primary melanoma, and metastatic melanoma. Real-time PCR (RT-PCR) and Western blotting were employed to quantify SPP1 expression in melanoma cells and tissues. Cell proliferation, wound healing, and Transwell assays were carried out to evaluate the effects of SPP1 and BET inhibitors in melanoma cells in vitro . A xenograft mouse model was used to investigate the effect of SPP1 and BET inhibitors on melanoma in vivo . Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the regulatory mechanism of BET inhibitors on SPP1. Results: SPP1 was identified as a melanoma driver by bioinformatics analysis, and meta-analysis determined it to be a diagnostic and prognostic biomarker for melanoma. SPP1 overexpression was associated with poor melanoma prognosis, and silencing SPP1 suppressed melanoma cell proliferation, migration, and invasion. Through a pilot drug screen, we identified BET inhibitors as ideal therapeutic agents that suppressed SPP1 expression. Also, SPP1 overexpression could partially reverse the suppressive effect of BET inhibitors on melanoma. We further demonstrated that bromodomain-containing 4 (BRD4) regulated SPP1 expression. Notably, BRD4 did not bind directly to the SPP1 promoter but regulated SPP1 expression through NFKB2. Silencing of NFKB2 resembled the phenotype of BET inhibitors treatment and SPP1 silencing in melanoma. Conclusion: Our findings highlight SPP1 as an essential target of BET inhibitors and provide a novel mechanism by which BET inhibitors suppress melanoma progression via the noncanonical NF-κB/SPP1 pathway., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Myeloid-derived suppressor cells (MDSCs) have become increasingly recognized as facilitators of tumor development. However, the role of MDSCs in papillary thyroid carcinoma (PTC) progression has not been clearly explored., Objective: We aimed to evaluate the levels and function of circulating MDSCs in PTC., Methods: The proportion of circulating polymorphonuclear (PMN)-MDSCs and mononuclear-MDSCs from patients with PTC or benign thyroid nodules and healthy controls was measured using flow cytometry. For immunosuppressive activity analysis, sorted circulating MDSCs were cocultured with CD3/CD28-costimulated T lymphocytes and the proliferation of T cells was determined. PTC cell lines (TPC-1 and BC-PAP) were cocultured with PMN-MDSCs, and the effects on cell migration, invasion, proliferation, and apoptosis were evaluated. The differential expressed microribonucleic acids (RNAs) and messenger RNAs and their function were also explored in TPC-1 cells cocultured with or without PMN-MDSCs., Results: PMN-MDSCs were increased in peripheral blood mononuclear cells of patients with PTC. Circulating PMN-MDSCs displayed strong T cell suppressive activity. PTC cells demonstrated enhanced invasive capabilities in vitro and in vivo when cocultured with sorted PMN-MDSCs. PMN-MDSCs decreased expression of miR-486-3p and activated nuclear factor kappa B2 (NF-κB2), a direct target of miR-486-3p. Rescue of miR-486-3p diminished the cell migration and invasion induced by PMN-MDSCs., Conclusion: Collectively, our work indicates that circulating PMN-MDSCs promote PTC progression. By suppressing miR-486-3p, PMN-MDSCs promote the activity of the NF-κB2 signaling pathway, resulting in accelerated invasion of PTC cells, which may provide new therapeutic strategies for treatment of thyroid cancer., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The BCR recognizes foreign Ags to initiate humoral immunity that needs isotype-switched Abs generated via class switch recombination (CSR); however, stimulating the BCR in the absence of costimulation (e.g., CD40) does not induce CSR; thus, it remains elusive whether and how the BCR induces CSR mechanistically. Autoreactive B cells can maintain anergy via unresponsiveness of their BCRs to self-antigens. However, it remains unknown what molecule(s) restrict BCR signaling strength for licensing BCR-induced CSR and whether deficiency of such molecule(s) disrupts autoreactive B cell anergy and causes B cell-mediated diseases by modulating BCR signaling. In this study, we employ mouse models to show that the BCR's capacity to induce CSR is restrained by B cell-intrinsic checkpoints TRAF3 and TRAF2, whose deletion in B cells enables the BCR to induce CSR in the absence of costimulation. TRAF3 deficiency permits BCR-induced CSR by elevating BCR-proximal signaling intensity. Furthermore, NF-κB2 is required for BCR-induced CSR in TRAF3-deficient B cells but not for CD40-induced or LPS-induced CSR, suggesting that TRAF3 restricts NF-κB2 activation to specifically limit the BCR's ability to induce CSR. TRAF3 deficiency also disrupts autoreactive B cell anergy by elevating calcium influx in response to BCR stimulation, leading to lymphoid organ disorders and autoimmune manifestations. We showed that TRAF3 deficiency-associated autoimmune phenotypes can be rectified by limiting BCR repertoires or attenuating BCR signaling strength. Thus, our studies highlight the importance of TRAF3-mediated restraint on BCR signaling strength for controlling CSR, B cell homeostasis, and B cell-mediated disorders., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Vaspin, visceral-adipose-tissue-derived serine protease inhibitor, is involved in the development of obesity, insulin resistance, inflammation, and energy metabolism. Our previous study showed vaspin expression and its regulation in the ovary; however, the role of this adipokine in ovarian cells has never been studied. Here, we studied the in vitro effect of vaspin on various kinase-signaling pathways: mitogen-activated kinase (MAP3/1), serine/threonine kinase (AKT), signal transducer and activator of transcription 3 (STAT3) protein kinase AMP (PRKAA1), protein kinase A (PKA), and on expression of nuclear factor kappa B (NFKB2) as well as on steroid synthesis by porcine ovarian cells. By using western blot, we found that vaspin (1 ng/ml), in a time-dependent manner, increased phosphorylation of MAP3/1, AKT, STAT3, PRKAA1, and PKA, while it decreased the expression of NFKB2. We observed that vaspin, in a dose-dependent manner, increased the basal steroid hormone secretion (progesterone and estradiol), mRNA and protein expression of steroid enzymes using real-time PCR and western blot, respectively, and the mRNA of gonadotropins (FSHR, LHCGR) and steroids (PGR, ESR2) receptors. The stimulatory effect of vaspin on basal steroidogenesis was reversed when ovarian cells were cultured in the presence of a PKA pharmacological inhibitor (KT5720) and when GRP78 receptor was knocked down (siRNA). However, in the presence of insulin-like growth factor type 1 and gonadotropins, vaspin reduced steroidogenesis. Thus, vaspin, by activation of various signaling pathways and stimulation of basal steroid production via GRP78 receptor and PKA, could be a new regulator of porcine ovarian function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1 -/- ) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34-66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

This study sought to evaluate the association of 28 single nucleotide polymorphisms (SNPs) within NFKB and inflammasome pathway genes with the risk of rheumatoid arthritis (RA) and response to TNF inhibitors (TNFi). We conducted a case-control study in a European population of 1194 RA patients and 1328 healthy controls. The association of potentially interesting markers was validated with data from the DANBIO (695 RA patients and 978 healthy controls) and DREAM (882 RA patients) registries. The meta-analysis of our data with those from the DANBIO registry confirmed that anti-citrullinated protein antibodies (ACPA)-positive subjects carrying the NFKB2 rs11574851T allele had a significantly increased risk of developing RA (PMeta&#95;ACPA + = 0.0006) whereas no significant effect was found in ACPA-negative individuals (PMeta&#95;ACPA- = 0.35). An ACPA-stratified haplotype analysis including both cohorts (n = 4210) confirmed that ACPA-positive subjects carrying the NFKB2 TT haplotype had an increased risk of RA (OR = 1.39, P = 0.0042) whereas no effect was found in ACPA-negative subjects (OR = 1.04, P = 0.82). The meta-analysis of our data with those from the DANBIO and DREAM registries also revealed a suggestive association of the NFKB2 rs1056890 SNP with larger changes in DAS28 (OR = 1.18, P = 0.007). Functional experiments showed that peripheral blood mononuclear cells from carriers of the NFKB2 rs1005044C allele (in LD with the rs1056890, r2 = 1.00) showed increased production of IL10 after stimulation with LPS (P = 0.0026). These results provide first evidence of a role of the NFKB2 locus in modulating the risk of RA in an ACPA-dependent manner and suggest its implication in determining the response to TNFi. Additional studies are now warranted to further validate these findings.

Gastric cancer is one of the most common worldwide types of cancer. It is a multifactorial disease and both environmental and genetic factors play an important role in its etiology. Evaluation of the relative expression level of NFKB2 gene in two groups of patients: peptic ulcer and gastric cancer and its role in the pathomechanism of these diseases was the aim of this study. RNA was isolated from: 79 samples of peptic ulcer, 22 gastric cancer and 11 control tissue. The real-time PCR technique was used to study the expression of NFKB2 gene. The relative expression level of NFKB2 gene was a variable in all three studied groups. The relative NFKB2 gene expression depends on the type of a disease. Peptic ulcer cases showed the increased relative NFKB2 gene expression to control group (p = 0.0000). Cancer cases presented decreased relative NFKB2 gene expression to normal stomach tissue (p = 0.0183). There are statistically important differences in the investigated gene expression between peptic ulcer, where the expression level is higher comparing to gastric cancer and control tissue which confirmed that such an activation is connected with an inflammatory process. The relative expression level of NFKB2 is decreased in cancer cases as opposed to control tissue and peptic ulcer cases which could suggest that during carcinogenesis of gastric cancer inhibition of NF-kB pathway takes place which could be a promising factor for patients.

Mutations affecting the non-canonical pathway of NF-κB were recently identified to underlie a form of common variable immunodeficiency strongly associated with autoimmunity. Although intrinsic B-cell abnormalities explain most of the humoral defects of this disease, detailed data on the impact of NFKB2 on follicular helper (Tfh) and regulatory (Tregs) T cells are scarce. Here, we show that Tfh, CXCR5 + , and CXCR5 - Treg cell subsets were significantly reduced in patients heterozygous for a truncating mutation of NFKB2. Plasma CXCL13 levels were reduced, underlining an important role for NFKB2 in regulating the germinal center (GC) response. Proinflammatory IFNγ, IL-17 and IL-10 cytokine production by CD4 T cells was lower in the mutated patients, but the production of IL-4 and IL-21 was not altered. Taken together, our findings show that NFKB2 influences the quality and efficiency of human GC reaction, by affecting not only the B cells but also GC-relevant T cell subsets., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Non-canonical NFκB (NC-NFκB) pathway plays an influential role in metastasis, which promotes cancer proliferation and progression. The aim of the study was to examine the expression of NC-NFκB proteins and their correlation with clinicopathological factors associated with metastatic cases of uveal melanoma (UM) and with the patient outcome., Method: Expression of NC-NFκB proteins (p52, RelB, and co-expression of p52/RelB) was evaluated in 75 formalin-fixed cases of uveal melanoma by immunohistochemistry. Validation of nuclear immunoreactivity was done by western blotting. Transcriptional status of NC-NFκB genes was assessed in 60 fresh tumor tissues by quantitative real-time PCR. Co-immunoprecipitation was performed to determine the presence of native p52/RelB heterodimer in UM. Prognostic relevance was determined using Cox proportional hazard and Kaplan-Meier methods., Results: Immunohistochemical expression of p52, RelB, and their co-expression was observed in 81%, 68.7%, 56.2% of metastatic cases, respectively, while their expression was seen only in 38%, 33% and 30% of non-metastatic cases. Loss of BAP-1 was correlated with expression of p52 and RelB proteins. Co-immunoprecipitation assay confirmed the putative interaction of p52 with RelB protein in metastatic cases of uveal melanoma. Co-expression of p52/RelB and expression of p52 protein was significantly correlated with decreased metastasis-free survival (MFS) (p = 0.004; p = 0.002) and overall survival (OS) (p = 0.004; p = 0.032), while the RelB expression only correlated with reduced MFS (p = 0.003)., Conclusion: Our data showed that non-canonical NFκB proteins were significantly higher in metastatic cases and associated with poor outcome of the patients. Furthermore, the p52 protein could be used as a potential therapeutic biomarker for metastatic cases in uveal melanoma.

Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying proteins that facilitate these changes are poorly understood. The histone demethylase LSD1 is an epigenetic modifier that promotes plasmablast formation, but its role in B cell development has not been explored. In this study, a role for LSD1 in the development of B cell subsets was examined. B cell-conditional deletion of LSD1 in mice resulted in a decrease in MZB whereas follicular B cells and bone marrow B cell populations were minimally affected. LSD1 repressed genes in MZB that were normally upregulated in the myeloid and follicular B cell lineages. Correspondingly, LSD1 regulated chromatin accessibility at the motifs of transcription factors known to regulate splenic B cell development, including NF-κB motifs. The importance of NF-κB signaling was examined through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-κB-inhibited transitional B cells failed to undergo full MZB development. Gene expression and chromatin accessibility analyses of in vivo- and ex vivo-generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of noncanonical NF-κB signaling. Additionally LSD1 was found to interact with the noncanonical NF-κB transcription factor p52. Together, these data reveal that the epigenetic modulation of the noncanonical NF-κB signaling pathway by LSD1 is an essential process during the development of MZB., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Glucagon promotes hepatic gluconeogenesis and maintains whole-body glucose levels during fasting. The regulatory factors that are involved in fasting glucagon response are not well understood. Here we report a role of p52, a key activator of the noncanonical nuclear factor-kappaB signaling, in hepatic glucagon response. We show that p52 is activated in livers of HFD-fed and glucagon-challenged mice. Knockdown of p52 lowers glucagon-stimulated hyperglycemia, while p52 overexpression augments glucagon response. Mechanistically, p52 binds to phosphodiesterase 4B promoter to inhibit its transcription and promotes cAMP accumulation, thus augmenting the glucagon response through cAMP/PKA signaling. The anti-diabetic drug metformin and ginsenoside Rb1 lower blood glucose at least in part by inhibiting p52 activation. Our findings reveal that p52 mediates glucagon-triggered hepatic gluconeogenesis and suggests that pharmacological intervention to prevent p52 processing is a potential therapeutic strategy for diabetes.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Genetic studies are identifying an increasing number of monogenic causes of Common Variable Immunodeficiency (CVID). Pathogenic variants in the C-terminus of NFKB2 have been identified in the subset of CVID patients whose immunodeficiency is associated with ectodermal dysplasia and central adrenal insufficiency. We describe 2 unrelated CVID pedigrees with 4 cases of pathogenic stop gain variants (c.1903C > T) in the ankyrin repeat domain (ARD) of NF-κB2, leading to a premature truncation of the protein at p.Arg635Term (R635X). By immunophenotyping and functional ex vivo B- and T-cell experiments we characterized the variant by reduced class-switched memory B-cell counts and immature plasmablasts, unable to produce IgG and IgA. Features of a poor proliferative T-cell response and reduced expansion of CD4 + CXCR5 + T cells was only observed in the two clinically affected index cases without any clear clinical correlate. In conclusion, pathogenic stop variants in the ARD of NFKB2 can cause 'infection-only' CVID with an abnormal B-cell phenotype and a variable clinical penetrance., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aim: Although genetic factors are presumed to account only for a part of the inter-individual variation in lung cancer susceptibility, the results are conflicting and there are no data available regarding the Polish population. We, therefore, performed a case-control study to investigate the association of seven selected single nucleotide polymorphisms (SNPs), in genes coding for excision repair cross-complimentary group 1 (ERCC1: rs11615, rs3212986, rs2298881), nuclear factor ĸB (NFKB2: rs7897947, rs12769316), bone morphogenetic protein 4 (BMP4: rs1957860), complement receptor 1 (CR1: rs7525160) and del/ins polymorphism in the family hypoxia inducible factor 2 gene (EGLN2: rs10680577), with non-small cell lung cancer (NSCLC) risk., Materials and Methods: Real-time PCR with melting curve analysis was used for genotyping of NSCLC patients and healthy individuals of Polish origin., Results: The ERCC1 rs11615 T allele and rs3212986 GG homozygosity were found to be associated with a higher risk of developing NSCLC. In addition, NFKB2 rs12769316 GG homozygosity was more frequently detected among male patients than controls, while no significant differences were found between the five polymorphisms., Conclusion: ERCC1 polymorphisms may affect NSCLC risk in the Polish population, while the NFKB2 variant may be a possible marker of the disease in males., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings.

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiβ mRNA stability and expression. Positively regulation of rhogdiβ mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiβ mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIβ axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target., (© 2019 Wiley Periodicals, Inc.)

Common variable immunodeficiency is the most prevalent of the primary immunodeficiency diseases, yet its pathogenesis is largely poorly understood. Of the cases that are monogenic, many arise due to pathogenic variants in NFKB1 and NFKB2. Here, we report enteroviral encephalomyelitis as the cause of a fatal neurodegenerative condition in a patient with a novel heterozygous mutation in NFKB2 (c.2543insG, p.P850Sfs36*) that disrupts non-canonical NF-κB signaling. Investigations of primary and secondary lymphoid tissue demonstrated a complete absence of B cells and germinal centers. Despite multiple negative viral PCR testing of cerebrospinal fluid during her disease progression, post-mortem analysis of cerebral tissue revealed a chronic lymphocytic meningoencephalitis, in the presence of Cocksackie A16 virus, as the cause of death. The clinical features, and progression of disease reported here, demonstrate divergent clinical and immunological phenotypes of individuals within a single family. This is the first reported case of fatal enteroviral encephalomyelitis in a patient with NF-κB2 deficiency and mandates a low threshold for early brain biopsy and the administration of increased immunoglobulin replacement in any patient with a defect in this pathway and deterioration of neurological status.

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in NFKB2 have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous NFKB2 -mutations including eight patients with the common p.Arg853 * nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published NFKB2 -cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4 + or CD8 + T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of NFKB2 -associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in NFKB2 represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency.

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA)., Competing Interests: Author PAG received funding from Debiopharm International. Authors NW and GV are employees of Debiopharm International. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Muscle atrophy with aging is closely associated with chronic systemic inflammation and lifestyle-related diseases. Here, we assessed whether dried tofu intake during 5-month interval walking training (IWT) enhanced increases in thigh muscle mass and strength and ameliorated susceptibility to inflammation in older women. Subjects (n = 32, ~ 65 years) who performed IWT for > 6 months participated in this study. They were randomly divided into 2 groups: IWT + placebo intake (PLG, n = 16; 108 kcal, 0.2 g protein, 5.5 g fat, and 14.4 g carbohydrate) and IWT + dried tofu intake (DTG, n = 16; 111 kcal, 9.6 g protein, 6.0 g fat, and 4.6 g carbohydrate). They were instructed to repeat ≥ 5 sets of fast and slow walking for 3 min each at ≥ 70 and 40% peak aerobic capacity for walking, respectively, per day for ≥ 4 days/week. Immediately after daily exercise, subjects were instructed to consume the supplements assigned to each group. In the DTG, after IWT, the methylation increased at 4/6 sites in the promoter region of the NFKB2 gene in the whole blood (all, P < 0.04), with an 18% increase in the average methylation of the 6 sites (P = 0.035). On the other hand, in the PLG, the increase occurred at only 2/6 sites, with no significant increase in the average methylation of the 6 sites. No significant differences were observed in increases in thigh muscle strength or cross-sectional area between the groups (all, P > 0.2). Altogether, dried tofu supplementation during IWT likely enhanced the methylation of the NFKB2 gene more than IWT alone, without detectably enhanced increases in thigh muscle strength or cross-sectional area.

Niemann-Pick type C2 (NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of low-density lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2 (NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor (LTβR) activation or Baff receptor (BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system.

Objective: To investigate the clinical, immunological, and molecular manifestations of nuclear factor kappa-B subunit 2 (NFκB2) gene mutation associated common variable immunodeficiency (CVID) . Methods: A 14-month-old boy diagnosed with NFκB2-mutated CVID was admitted into Children's Hospital of Chongqing Medical University in December 2015. The clinical manifestations, biochemical tests, immunological function, molecular features, treatment, and follow-up of the patient were analyzed. The Chinese and PUBMED databases were searched with the key words "NFκB2" and "immune deficiency" and related literatures were reviewed. Results: The patient had 4 episodes of pneumonias and one otitis media since the age of 6 months. The serum immunoglobulin levels were IgG 2.73 g/L, IgA<0.07 g/L, and IgM 0.12 g/L. The percentage of peripheral lymphocyte subsets demonstrated increased CD3(+)T lymphocyte (81.8%), increased CD4(+) naïve T cell (39.1%), normal B cell (14.1%), low switched memory B and plasmablast B (respectively 0.1% and 0), and lightly diminished natural killer(NK) cell (4.13%). Within the peripheral CD4(+)T cells, the percentage of regulatory T cells (1.49% (control 4.08%)), T follicular helper (3.66% (control 11.0%)), and T helper 17 (9.65% (control 15.7%)) were decreased, while the percentage of T helper 2 (60.9% (control 46.5%)) was elevated. T lymphocyte proliferative response and T cell receptor repertoire diversity were normal. NK-cell cytotoxic activity was impaired. The whole-exome sequencing harbored a de novo heterozygous nonsense mutation in exon 22 (c.2557C>T; p. Arg853X) in the C-terminus of NF-κB2. The western blotting confirmed the decreased expression of NF-κB2 (p52) protein. The patient received intravenous immunoglobulin infusion monthly (400-600 mg/kg), followed by improvement of pulmonary infection. After searching the databases, a total of 28 cases (1 Chinese and 27 non-Chinese) were identified. There were 12 cases of nonsense mutation (5 were gain-of-function mutation), and 8 cases of missense and frameshift mutations, respectively. The main clinical manifestation was respiratory infection, followed by autoimmune diseases such as alopecia and trachyonychia. Fifteen cases developed adrenocorticotrophic hormone (ACTH) deficiency. Conclusions: NF-κB2 signaling pathway played an important role in T and B lymphocyte differentiation, and NK-cell cytotoxic activity. NFκB2 mutation should be considered in cases with recurrent infections, hypogammaglobulinemia, and decreased memory B cells and plasma cells, especially when combined with ACTH deficiency.