Canonical NF-κB Pathway Research Articles

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus induces early TRAF6-mediated NF-κB and IRF7 signaling pathways through TLRs

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.

Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response

ABSTRACT Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKβ phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.

RF04 | PSUN163 In High Glucose Exposed Endothelial Cells BTK is a Novel Component of the Nuclear Factor-Kappa B Pathway

Abstract Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFkB signaling in ischemic endothelial cells (EC). We have shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFkB pathway through activation of protein kinase C β (PKC β). However, the signaling pathways upstream of PKC β involved in this high glucose induced desensitization of NFkB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFkB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spot by the signal intensity of the corresponding non-phosphorylated spot for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton's tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling but not known to be expressed in ECs or play a role in EC function. Here we confirmed BTK expression in ECs by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid or by shRNA mediated knockdown decreased PKCβ-S661 phosphorylation and restored NFkB activation in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as a potentially new component of the NFkB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

B Cell Activation Results in IKK-Dependent, but Not c-Rel- or RelA-Dependent, Decreases in Transcription of the B Cell Tolerance-Inducing Gene Ets1.

Ets1 is a key transcription factor in B cells that is required to prevent premature differentiation into Ab-secreting cells. Previously, we showed that BCR and TLR signaling downregulate Ets1 levels and that the kinases PI3K, Btk, IKK, and JNK are required for this process. PI3K is important in activating Btk by generating the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate, to which Btk binds via its PH domain. Btk in turn is important in activating the IKK kinase pathway, which it does by activating phospholipase Cγ2→protein kinase Cβ signaling. In this study, we have further investigated the pathways regulating Ets1 in mouse B cells. Although IKK is well known for its role in activating the canonical NF-κB pathway, IKK-mediated downregulation of Ets1 does not require either RelA or c-Rel. We also examined the potential roles of two other IKK targets that are not part of the NF-κB signaling pathway, Foxo3a and mTORC2, in regulating Ets1. We find that loss of Foxo3a or inhibition of mTORC2 does not block BCR-induced Ets1 downregulation. Therefore, these two pathways are not key IKK targets, implicating other as yet undefined IKK targets to play a role in this process.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1−/−-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

Mechanisms underlying synergism between circularized tumor necrosis factor-related apoptosis inducing ligand and bortezomib in bortezomib-sensitive or -resistant myeloma cells.

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor-related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib-resistant U266 cells were exposed to low nanomolar concentrations of bortezomib±CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ-naïve and -resistant cells. The regimen up-regulated DR4 receptor internalization in MM cells, known to modulate both NF-κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non-canonical NF-κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up-regulation, NF-κB inducing kinase down-regulation, diminished p52 and p50 processing, and B-cell lymphoma-extra large (BCL-XL) down-regulation, but failed to inactivate the canonical NF-κB pathway, reflected by unchanged or increased expression of phospho-p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock-down, dominant-negative Fas-associated protein with death domain, knock-down of caspase-8, BCL-2/BCL-XL, or exposure to a caspase-9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib-resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34+ hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient-derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non-canonical NF-κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors' Dysfunction.

Endometrosis is a frequently occurring disease decreasing mares’ fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

Pharmacogenetic analysis of canonical versus noncanonical pathway of NF-kB in Crohn's disease patients under anti-tumor necrosis factor-α treatment.

This study explores the potential of gene polymorphisms in the canonical and noncanonical NF-kB signaling pathway as a prediction biomarker of anti-tumor necrosis factor (TNF)α response in Crohn's patients. A total of 109 Greek patients with Crohn's disease (CD) were recruited, and the genotype of TLR2 rs3804099, LTA rs909253, TLR4 rs5030728, and MAP3K14/NIK rs7222094 single nucleotide polymorphisms was investigated for association with response to anti-TNFα therapy. Patient's response to therapy was based on the Crohn's Disease Activity Index, depicting the maximum response within 24 months after initiation of treatment. Seventy-three patients (66.7%) were classified as responders while 36 as nonresponders (33.3%). Comparing allelic frequencies between responders and nonresponders, the presence of TLR2 rs3804099 T allele was associated with nonresponse (P = 0.003), even after stratification by anti-TNFα drugs (infliximab: P = 0.032, adalimumab: P = 0.026). No other association was identified for the rest of the polymorphisms under study. Haplotype analysis further enhanced the association of rs3804099 T allele with loss of response, even though the results were NS (P = 0.073). Our results suggest that polymorphisms in the canonical NF-kB pathway genes could potentially act as a predictive biomarker of anti-TNFα response in CD.

The MIP-T3 from shrimp Litopenaeus vannamei restricts white spot syndrome virus infection via regulating NF-κB activation

In vertebrate, MIP-T3 (microtubule-interacting protein associated with TRAF3) functions as a regulator of innate immune response that involves many cellular processes. However, the immune response regulated by shrimp (an arthropod) MIP-T3 remains unrevealed. In the present study, a MIP-T3 homolog from shrimp Litopenaeus vannamei (named as LvMIP-T3) was cloned and identified. LvMIP-T3 had a 2076 bp open reading frame (ORF), encoding a polypeptide of 691 amino acids that contained a classic coiled-coil domain in the C-terminal that showed a high degree of conservation to other homologs. LvMIP-T3 could interact with LvTRAF6, a member of the canonical NF-κB pathway, but not LvTRAF3, which implies that LvMIP-T3 is able to regulate NF-κB activity via its interaction with LvTRAF6. In addition, LvMIP-T3 was substantially inducted in response to white spot syndrome virus (WSSV) challenge, and we demonstrated that LvMIP-T3 facilitated the expression of NF-κB-mediated several Penaeidins (antimicrobial peptides, AMPs) to oppose infection. Taken together, we identified a MIP-T3 homolog from shrimp L. vannamei that played a positive role in the TRAF6/NF-κB/AMPs axis mediated defense response, which will contribute to better understand the regulator relationship among members of the canonical NF-κB pathway in shrimp, and provides some insights into disease resistance breeding.

Changes Within H3K4me3-Marked Histone Reveal Molecular Background of Neutrophil Functional Plasticity.

Neutrophils are a heterogenous population capable of both antimicrobial functions and suppressor ones, however, no specific pattern of transcription factors controlling this plasticity has been identified. We observed rapid changes in the neutrophil status after stimulation with LPS, pre-activating concentration of TNF-α, or IL-10. Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3 allowed us to identify various transcriptional start sites (TSSs) associated with plasticity and heterogeneity of human neutrophils. Gene Ontology analysis demonstrated great variation within target genes responsible for neutrophil activation, cytokine production, apoptosis, histone remodelling as well as NF-κB transcription factor pathways. These data allowed us to assign specific target genes positioned by H3K4me3-marked histone with a different pattern of gene expression related to NF-κB pathways, apoptosis, and a specific profile of cytokines/chemokines/growth factors realised by neutrophils stimulated by LPS, IL-10, or TNF-α. We discovered IL-10-induced apoptotic neutrophils being transcriptionally active cells capable of switching the profile of cytokines/chemokines/growth factors desired in resolving inflammation via non-canonical NF-κB pathway with simultaneous inhibition of canonical NF-κB pathway. As apoptotic/suppressive neutrophils induced by IL-10 via positioning genes within H3K4me3-marked histone were transcriptionally active, newly described DNA binding sites can be considered as potential targets for immunotherapy.Graphical H3K4me3 histone ChIP-Seq analysis reveals molecular drivers critical for switching neutrophils from their pro- to anti-inflammatory properties.

Alphabetti kinase Spaghetti: the complex roles of IKKα and β in the canonical NF-κB pathway.

Numerous studies, published over many years, have established the key role that the IκB kinase (IKK) subunits, α and β, play in regulating the Nuclear Factor κB (NF-κB) pathway. This research generally concluded that their functions can be separated, with IKKβ being the critical regulator of the canonical NF-κB pathway, while IKKα functions as the key activating kinase for the non-canonical pathway. However, other roles for these kinases have been described and several reports concluded that this separation of their functions may not always be the case. This commentary discusses the recent report by Biochem J. 479, 305-325, who elegantly demonstrate that in KRAS driven colorectal cancer cell lines, IKKα is an important regulator of the canonical NF-κB pathway. As is so often the case with trying to understand the complexity of NF-κB signalling, cellular context is everything.

The antiviral role of NF-κB-mediated immune responses and their antagonism by viruses in insects.

The antiviral role of innate immune responses mediated by the NF-κB family of transcription factors is well established in vertebrates but was for a long time less clear in insects. Insects encode two canonical NF-κB pathways, the Toll and Imd ('immunodeficiency') pathways, which are best characterised for their role in antibacterial and antifungal defence. An increasing body of evidence has also implicated NF-κB-mediated innate immunity in antiviral responses against some, but not all, viruses. Specific pattern recognition receptors (PRRs) and molecular events leading to NF-κB activation by viral pathogen-associated molecular patterns (PAMPs) have been elucidated for a number of viruses and insect species. Particularly interesting are recent findings indicating that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway detects viral RNA to activate NF-κB-regulated gene expression. We summarise the literature on virus-NF-κB pathway interactions across the class Insecta, with a focus on the dipterans Drosophila melanogaster and Aedes aegypti. We discuss potential reasons for differences observed between different virus-host combinations, and highlight similarities and differences between cGAS-STING signalling in insects versus vertebrates. Finally, we summarise the increasing number of known molecular mechanisms by which viruses antagonise NF-κB responses, which suggest that NF-κB-mediated immunity exerts strong evolutionary pressures on viruses. These developments in our understanding of insect antiviral immunity have relevance to the large number of insect species that impact on humans through their transmission of human, livestock and plant diseases, exploitation as biotechnology platforms, and role as parasites, pollinators, livestock and pests.

Comparison of the Safety and Efficacy of Tofacitinib and Fingolimod in TNBS‐Induced Colitis Model in Adult Zebrafish: The Role of Myd88/NF‐κB/TNF‐α Signaling Pathway

Due to the inadequate response, side effects, parenteral route of administration, and immunogenicity, oral targeted small molecules seem to replace monoclonal antibodies in the future management of ulcerative colitis (UC). In order to pave the way for clinical translations of small molecules in UC, for the first time, we compared the efficacy of fingolimod and tofacitinib in preclinical model of colitis, and uncovered another mechanism of action that these medicines may act through.We implicated adult zebrafish to find out its pros/cons, investigated aquatic toxicity of the mentioned compounds, and designed a procedure to evaluate compound's efficacy in adult zebrafish colitis model; hence, facilitating screening of further agents.Tofacitinib and fingolimod were investigated to be in category 1 of Globally Harmonized System (GHS) aquatic toxicity classification with the LC50 of 0.9014 and 0.36 mg/L, respectively, based on the acute toxicity assessment within 96 h.Considering efficacy assessments, colitis was induced through intrarectal administration of 160 mM TNBS for 48 h. Then, animals were exposed to different concentrations of tofacitinib (0.25, 0.57, 0.72, 0.90 mg/L) and fingolimod (0.23, 0.28, 0.36, 0.45 mg/L) for 96 h. Subsequent analyses revealed that TNBS significantly reduced length of villi, number of goblet cells, level of TNF‐α, MyD88, and NF‐κB2, increased thickness of villi and necrosis, and induced histopathological changes, including intestinal edema, submucosal hemorrhage/hyperemia with the presence of inflammatory cells. All the aforementioned parameters were considerably enhanced almost dose‐dependently with both agents, wherein the effect of 0.45 mg/L fingolimod was superior to other groups. As well, there were no signs of hyperemia/hemorrhage in harvested intestinal samples. Resultantly, we revealed that these medications may suppress canonical and non‐canonical NF‐κB pathways through targeting toll‐like receptors and MyD88.Given the better efficacy and multi‐targeted action of fingolimod, it worth to focus on sphingosine‐1 phosphate receptor modulators in clinical trials of UC and compare them with other classes of medications; albeit, their ecotoxicity aspect should not be overlooked.

Characterization of An Inducible p65 Knockout Mouse Model for Studying Glioblastoma

Glioblastoma (GBM) is a malignant brain cancer that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor promoting M2 phenotype which encourages tumor growth. However, there are limited studies about the specific effect(s) that microglia have on GBM thus requiring further research into how these cells affect GBM tumors.Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor‐associated cells that are involved in sustaining GBM tumors could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor‐kappa B (NF‐κB) pathway, which has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on characterization of the p65fl/fl/CX3CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF‐κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF‐κB pathway, should be deleted in microglia due to the C‐X3‐C chemokine receptor 1 (CX3CR1) promoter. Tamoxifen binds to the estrogen receptor on the cre recombinase (creER) so that the cre recombinase can recombine the floxed p65 (p65fl/fl) gene in microglia. Characterization of this particular mouse model is necessary to determine if the floxed p65 gene is effectively deleted in microglia from these mice. Liquid chromatography‐mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen‐treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the floxed p65 gene in isolated microglia compared to p65 knockout mice given the vehicle control. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl/CX3CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggested that the floxed p65 gene is partially deleted in microglia of the tamoxifen‐treated p65 knockout mice compared to the vehicle control mice.

NF-κB-inducing kinase (NIK) is activated in pancreatic β-cells but does not contribute to the development of diabetes

The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in β-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic β-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous β-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic β-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of β-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes.

Dimethyl Fumarate Induces Apoptosis via Inhibiting NF-κB and STAT3 Signaling in Adult T-cell Leukemia/Lymphoma Cells

Adult T-cell leukemia (ATL) is a peripheral T lymphocytic malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Despite treatment that includes novel agents that have been developed, most of ATL patients relapse and acquire multidrug resistance. As a result, the creation of newer agents is critical. Dimethyl fumarate (DMF) has several effects in cancer cells, including cell signaling, proliferation and cell death. However, its antitumor effects on ATL cells remain unknown. In this study, we looked at DMF's antitumor effects on ATL cells. We examined the effects of DMF on proliferation and apoptosis using the trypan blue exclusion assay and annexin V/propidium iodide staining in HTLV-1-infected and transformed T-cell lines, MT-1 and MT-2 cells. We also evaluated the effects of DMF on the nuclear factor-kappa B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) signaling pathways and anti-apoptotic proteins by immunoblotting. DMF inhibited proliferation and induced apoptosis in MT-1 and MT-2 cells by activating poly ADP-ribose polymerase (PARP). Furthermore, DMF inhibited the constitutive activation of both canonical and non-canonical NF-κB pathways in MT-2 cells and the non-canonical NF-κB pathway in MT-1 cells. DMF also inhibited the constitutive tyrosine phosphorylation of STAT3 and the expression of anti-apoptotic proteins, c-IAP2 and survivin in both cells. These results indicate that DMF inhibits proliferation and induces apoptosis in HTLV-1-infected and transformed T-cells by suppressing NF-κB and STAT3 signaling pathways. DMF should be investigated further as a novel agent for ATL.

A genome-scale screen for synthetic drivers of T cell proliferation.

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cellfunctions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-β receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.

Plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects that prevents menopausal-related osteoporosis in mice

Osteoporosis is caused by enhanced bone resorption and relatively reduced bone formation. There is an unmet need to develop new agents with both antiresorptive and anabolic effects to treat osteoporosis, although drugs with either effect alone are available. A small molecular compound, plumbagin, was reported to inhibit receptor activator of nuclear factor kappa-B ligand–induced osteoclast (OC) differentiation by inhibiting IκBα phosphorylation–mediated canonical NF-κB activation. However, the key transcriptional factor RelA/p65 in canonical NF-κB pathway functions to promote OC precursor survival but not terminal OC differentiation. Here, we found that plumbagin inhibited the activity of NF-κB inducing kinase, the key molecule that controls noncanonical NF-κB signaling, in an ATP/ADP-based kinase assay. Consistent with this, plumbagin inhibited processing of NF-κB2 p100 to p52 in the progenitor cells of both OCs and osteoblasts (OBs). Interestingly, plumbagin not only inhibited OC but also stimulated OB differentiation in vitro. Importantly, plumbagin prevented trabecular bone loss in ovariectomized mice. This was associated with decreased OC surfaces on trabecular surface and increased parameters of OBs, including OB surface on trabecular surface, bone formation rate, and level of serum osteocalcin, compared to vehicle-treated mice. In summary, we conclude that plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects on bone and could represent a new class of agent for the prevention and treatment of osteoporosis.

Superoxide Dismutase-3 Downregulates Laminin α5 Expression in Tumor Endothelial Cells via the Inhibition of Nuclear Factor Kappa B Signaling.

Simple SummaryTumor-infiltrating lymphocytes determine cancer prognosis and the response to immunotherapy. The balance of laminin-α4/laminin-α5 isoforms in the endothelial basement membrane plays an instructive function in regulating the diapedesis of immune cells under inflammatory conditions. Previous studies showed that the extracellular superoxide dismutase (SOD)3 induces laminin-α4, which is associated with improved disease-free survival of colorectal cancer patients. The aim of our study was to determine whether SOD3 also affects the expression of laminin-α5 in the tumor vasculature. The results showed that SOD3 differentially regulates laminin-α4 and laminin-α5 in the tumor endothelium. SOD3 promoted notable transcriptomic changes in tumor-stimulated endothelial cells, including the inhibition of the nuclear factor kappa B (NF-κB) pathway, an inductor of laminin α5 transcription. Therefore, high SOD3 levels in the tumor vasculature shifted the laminin α4/α5 balance towards the laminin-α4high/laminin-α5low phenotype, which is permissive for T cell diapedesis into tumors and explains the improved cancer immune surveillance associated to high SOD3 levels.The balance between laminin isoforms containing the α5 or the α4 chain in the endothelial basement membrane determines the site of leukocyte diapedesis under inflammatory conditions. Extracellular superoxide dismutase (SOD3) induces laminin α4 expression in tumor blood vessels, which is associated with enhanced intratumor T cell infiltration in primary human cancers. We show now that SOD3 overexpression in neoplastic and endothelial cells (ECs) reduces laminin α5 in tumor blood vessels. SOD3 represses the laminin α5 gene (LAMA5), but LAMA5 expression is not changed in SOD1-overexpressing cells. Transcriptomic analyses revealed SOD3 overexpression to change the transcription of 1682 genes in ECs, with the canonical and non-canonical NF-κB pathways as the major SOD3 targets. Indeed, SOD3 reduced the transcription of well-known NF-κB target genes as well as NF-κB-driven promoter activity in ECs stimulated with tumor necrosis factor (TNF)-α, an NF-κB signaling inducer. SOD3 inhibited the phosphorylation and degradation of IκBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor alpha), an NF-κB inhibitor. Finally, TNF-α was found to be a transcriptional activator of LAMA5 but not of LAMA4; LAMA5 induction was prevented by SOD3. In conclusion, SOD3 is a major regulator of laminin balance in the basement membrane of tumor ECs, with potential implications for immune cell infiltration into tumors.

CD40L-expressing CD4+ T cells prime adipose-derived stromal cells to produce inflammatory chemokines

The therapeutic potential of culture-adapted adipose-derived stromal cells (ASCs) is largely related to their production of immunosuppressive factors that are inducible in vitro by priming with inflammatory stimuli, in particular tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). In vivo, obesity is associated with chronic inflammation of white adipose tissue, including accumulation of neutrophils, infiltration by IFNγ/TNFα-producing immune cells, and ASC dysfunction. In the current study, we identified in obese patients a simultaneous upregulation of CD40Lin the adipose tissue stroma vascular fraction (AT-SVF), correlated with the Th1 gene signature, and an overexpression of CD40 by native ASCs. Moreover, activated CD4+ T cells upregulated CD40 on culture-expanded ASCs and triggered their production of IL-8 in a CD40L-dependent manner, leading to an increased capacity to recruit neutrophils. Finally, activation of ASCs by sCD40L or CD40L-expressing CD4+ T cells relies on both canonical and non-canonical NF-κB pathways, and IL-8 was found to be coregulated with NF-κB family members in AT-SVF. These data identify the CD40-CD40L axis as a priming mechanism of ASCs, able to modulate their cross talk with neutrophils in an inflammatory context, and their functional capacity for therapeutic applications.