NF-kB Activation Research Articles

CARD11 regulates the thymic Treg development in an NF-κB-independent manner.

CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner. We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Tobacco smoke condensate-induced senescence in endothelial cells was ameliorated by colchicine treatment via suppression of NF-κB and MAPKs P38 and ERK pathways activation

Smoking is the major cause of cardiovascular diseases and cancer. It induces oxidative stress, leading to DNA damage and cellular senescence. Senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase, which are known to play a vital role in the initiation and progression of cardiovascular diseases and metastasis in cancer. The current study investigated the smoking induced cellular senescence and employed colchicine that blocked senescence in endothelial cells exposed to tobacco smoke condensate. Colchicine prevented oxidative stress and DNA damage in tobacco smoke-condensate-treated endothelial cells. Colchicin reduced β-gal activity, improved Lamin B1, and attenuated cell growth arrest markers P21 and P53. Colchicine also ameliorated the expression of SASP factors and inhibited the activation of NF-kB and MAPKs P38 and ERK. In summary, colchicine inhibited tobacco smoke condensate-induced senescence in endothelial cells by blocking the activation of NF-kB and MAPKs P38 and ERK.Graphical

Okadaic acid enhances NfKB, TLR-4, caspase 3, ERK ½, c-FOS, and 8-OHdG signaling pathways activation in brain tissues of zebrafish larvae

This study was designed to investigate the potential neuronal damage mechanism of the okadaic acid (OA) in the brain tissues of zebrafish embryos by evaluating in terms of immunofluorescence of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG signaling pathways. We also evaluated body malformations. For this purpose, zebrafish embryos were exposed to 0.5 μg/ml, 1 μg/ml and 2.5 μg/ml of OA for 5 days. After application, FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG respectively. The results indicated that OA caused immunofluorescence positivity of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG in a dose-dependent manner in the brain tissues of zebrafish embryos. Pericardial edema (PE), nutrient sac edema (YSE) and body malformations, tail malformation, short tail and head malformation (BM) were detected in zebrafish embryos. These results suggest that OA induces neuronal damage by affecting the modulation of DNA damage, apoptotic, and inflammatory activities in the brain tissues of zebrafish embryos. The increase in signaling pathways shows that OA can cause damage in the structure and function of brain nerve cells. Our results provide a new basis for the comprehensive assessment of the neural damage of OA and will offer enable us to better understand molecular the mechanisms underlying the pathophysiology of OA toxicity.

Geraniin Alleviates Inflammation in Caco-2 Cells and Dextran Sulfate Sodium-Induced Colitis Mice by Targeting IL-1β.

IL-1β is an important cytokine implicated in the progression of inflammatory bowel disease (IBD) and intestinal barrier dysfunction. The polyphenolic compound, geraniin, possesses bioactive properties, such as antitumor, antioxidant, anti-inflammatory, antihypertensive, and antiviral activities; however, its IL-1β-targeted anticolitis activity remains unclear. Here, we evaluated the inhibitory effect of geraniin in IL-1β-stimulated Caco-2 cells and a dextran sulfate sodium (DSS)-induced colitis mouse model. Geraniin blocked the interaction between IL-1β and IL-1R by directly binding to IL-1β and inhibited the IL-1β activity. It suppressed IL-1β-induced intestinal tight junction damage in human Caco-2 cells by inhibiting IL-1β-mediated MAPK, NF-kB, and MLC activation. Moreover, geraniin administration effectively reduced colitis symptoms and attenuated intestinal barrier injury in mice by suppressing elevated intestinal permeability and restoring tight junction protein expression through the inhibition of MAPK, NF-kB, and MLC activation. Thus, geraniin exhibits anti-IL-1β activity and anticolitis effect by hindering the IL-1β and IL-1R interaction and may be a promising therapeutic anti-IL-1β agent for IBD treatment.

Comparative response to PDT with methyl-aminolevulinate and temoporfin in cutaneous and oral squamous cell carcinoma cells

Cutaneous and Head and Neck squamous cell carcinoma (CSCC, HNSCC) are among the most prevalent cancers. Both types of cancer can be treated with photodynamic therapy (PDT) by using the photosensitizer Temoporfin in HNSCC and the prodrug methyl-aminolevulinate (MAL) in CSCC. However, PDT is not always effective. Therefore, it is mandatory to correctly approach the therapy according to the characteristics of the tumour cells. For this reason, we have used cell lines of CSCC (A431 and SCC13) and HNSCC (HN5 and SCC9). The results obtained indicated that the better response to MAL-PDT was related to its localization in the plasma membrane (A431 and HN5 cells). However, with Temoporfin all cell lines showed lysosome localization, even the most sensitive ones (HN5). The expression of mesenchymal markers and migratory capacity was greater in HNSCC lines compared to CSCC, but no correlation with PDT response was observed. The translocation to the nucleus of β-catenin and GSK3β and the activation of NF-κβ is related to the poor response to PDT in the HNSCC lines. Therefore, we propose that intracellular localization of GSK3β could be a good marker of response to PDT in HNSCC. Although the molecular mechanism of response to PDT needs further elucidation, this work shows that the most MAL-resistant line of CSCC is more sensitive to Temoporfin.

Abstract 1432: Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma

Abstract Microglia are resident macrophages of the central nervous system (CNS) and have crucial roles in normal brain homeostasis, function, and immune response. Microglia play key roles in the healthy brain, ranging from phagocytosis of dying neurons and CNS debris to modulating adaptability. The role of microglia has been well documented but their specific contributions to the development and progression of cancers, such as glioblastoma, have recently garnered wide attention. The zebrafish have proven novel tool for modeling innate immune responses. A macrophage-specific marker mpeg1 and its promoter are used in mpeg1-driven transgenes. mpeg1-driven transgenes mpeg1:mCherryFP are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. These zebrafish transgenes contribute to the fields of inflammation, infection, and leukocyte biology. We have recently developed a novel transparent transgenic homozygous strain Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) of zebrafish to the in-vivo study of NF-kB activity under normal and inflammation stage at the cellular, organ-specific and whole organism level. To perform an in-vivo study of microglial function, we are developing Casper;mpeg1:mCherryFP microglia specific strain and integrating with newly developed Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) strain through crossbreeding of Casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Casper;mpeg1:mCherryFP and Tg(6xNF-kB:EGFP);Casper. The presence of fluorescence mCherry and GFP, in crossbred progeny was identified through the screening of larvae at 72 HPF stage. The expression of these fluorescent proteins determines the presence of the reporter genes. F1 generation confirmed heterozygous and allowed them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4, and F5 generation until achieving homozygous progeny. This model allows for in-vivo examination of microglia functional morphology by using fluorescent and confocal microscopy. This novel transgenic model will be applied to identify novel anti-inflammatory drug inhibitors in NF-kB pathophysiology for glioblastoma and cardio-oncology experimental therapeutics. Citation Format: Surendra K. Rajpurohit, Sai Nasanally, Simran Hotwani, Taitum Gossman, Keshu Bhatt, Aiden Van Derhei, Rohan Pasi, Devan-Anmol Manhiani, Kirk Kimmerling, Balakrishna L. Lokeshwar. Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1432.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7586.

Abstract 3787: Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion

Abstract Background: Glutathione peroxidases(GPXs) are member of an antioxidant enzyme family. Their role is to eliminate the reactive oxygen species(ROS). Many available studies confirm that over expression of GPXs can promote cancer cell proliferation and invasion. Among the 8 GPXs, Glutathione peroxidase-1 (GPX1) is the most plentiful. Overexpression of GPX1 has been noted to promote invasion, migration, cisplatin resistance and proliferation in breast, colorectal and lung cancer. NF-kB is a ubiquitous nuclear transcription factor in the human body. that key role in cell proliferation, apoptosis and tumor progression. In reliable studies, inhibition of NF-kB expression has been shown to reduce the malignancy of Esophageal squamous cell carcinoma. Accordingly, the purpose of this study is to identify the GPX1 and NF-kB signaling pathway and their correlation with cancer cell proliferation and invasion in vitro using gastric cancer cell. Methods: In our study, we used cell culture, Northern blotting, cDNA microarray analysis, western blotting, RT-PCR, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, GPX1 knock-down with short hairpin RNS(shRNA), Standard two-chamber invasion assay, Chromatin immunoprecipitation assay. Results: First, we confirmed that the expression level of GPX1 was up-regulated by HGF(hepatocyte growth factor) in gastric cancer cells. The cells were treated with NF-kB inhibitor, Pyrrolidine dithiocarbamate(PDTC), and analyzed by Western blotting to identify the associated pathway of HGF-induced GPX1 with NF-kB. The HGF-induced GPX1 protein level was down regulated by PDTC. The role of GPX1 associated with NF-kB was determined by knock down GPX1 expression using GPX1-sh RNA. The GPX1-sh RNA treated cells showedvdown regulation of NF-kB and urokinase Plasminogen activator(uPA) also. To fine the upper regulator of GPX1 signaling pathway, we treated AKT inhibitor(LY294002) ant then analyzed by Western blotting to identify associated pathway of HGF-induced GPX1 with AKT. When AKT pathway was inhibited, GPX1 and NF-kB were down-regulated in gastric cancer cell. HGF-mediated cell proliferation and invasion in vitro was decreased in GPX1 knock down cell. We identified the putative binding site of GPX1 promotor containing NF-kB binding site and confirmed the function by CHIP assay. Conclusions: This study aimed to investigate relationship between GPX1 and NF-kB in gastric cancer cells. Results showed that HGF induced GPX1 expression through the NF-kB and AKT pathway and GPX1 may play a significant role in cell proliferation and invasion in gastric cancer. Therefore, GPX1 can be a potential therapeutic target of gastric cancer for the treatment. Citation Format: Byeong Il Jang, Ji Yoon Jung, Sung Ae Koh, Kyung Hee Lee. Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3787.

Abstract 411: PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells

Abstract Background: Chromosomal instability (CIN) frequently exerts innate immune response, being expected to sensitize immunotherapy. cGAS-STING pathway is a hub to regulate CIN-mediated innate immune activation. We previously demonstrated that small-molecule inhibitors targeting Centromere-associated Protein-E (CENP-E) profoundly accumulated CIN in cancer cells, leading to activation of cGAS-STING and its related innate immune pathways. In this study, aiming to deeply understand molecular mechanisms of CENP-E inhibitor (CENP-Ei)-induced CIN and innate immune activation, we conducted large-scale chemical screening with ~1,400 kinase inhibitors in CENP-Ei-treated reporter cells for NF-κB and IRF. We also experimentally validated the effects of hit compounds on CENP-Ei-induced innate immune activation. Material and Methods: GSK923295 and Cmpd-A were used as CENP-E inhibitors. HeLa, A549, A549 dual reporter cells were treated with the CENP-Ei at the indicated concentrations, subjected to immunofluorescent, transcriptome, FACS, reporter activity, or gene expression analyses. High-throughput screening (HTS) with ~1,400 kinase library was conducted in A549 dual reporter cells in combinational treatment with GSK923295. Combination effects of the representative hit compounds were confirmed in matrix-combination studies with the CENP-Ei. Results: Treatment with the CENP-Ei, Cmpd-A and GSK923295, induced CIN (e.g., multinucleation) in both A549 and A549 dual reporter cells, which elevated NFkB and IRF reporter activities as well as gene expressions of inflammatory cytokines and chemokines in innate immune pathways. Transcriptome analyses also confirmed that gene ontologies (GO) for innate immune response, such as cytosolic DNA sensing pathway and cytokine-cytokine receptor interaction pathway, were significantly enriched in CENP-Ei-treated CIN cells. Next, we conducted HTS of IRF-reporter activity with ~1,400 kinase library in combination with GSK923295 in A549 dual reporter cells. The HTS revealed that a series of inhibitors targeting PI3K-AKT-mTOR signaling pathways intensively suppressed CENP-Ei-induced IRF activation, which occupied ~40% of hit compounds. The matrix-combination studies confirmed that sapanisertib (TORC1/2 inhibitor) significantly attenuated CENP-Ei-induced IRF activity. Conclusions: CENP-Ei-induced CIN potently activates innate immune response pathways in cancer cells, in which PI3K-AKT-mTOR signaling pathway appears to be involved. We are initiating AI-based drug discovery to develop novel CENP-E inhibitors. Citation Format: Ryo Kamata, Hitoshi Saito, Yumi Hakozaki, Yukie Kashima, Gaku Yamamoto, Tomoko Yamamori Morita, Pinyi Lu, Akihiro Ohashi. PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 411.

Abstract 4264: The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment

Abstract High grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy but a majority relapse with chemoresistant disease. Research supports the notion that tumor-initiating cells (TICs), a subpopulation of drug resistant stem-like tumor cells, are responsible for facilitating relapse and therapies targeting these fecund cells may prolong remission. The tumor microenvironment (TME) plays a crucial role in therapy resistance, and tumor relapse. Cytotoxic chemotherapies have been shown to modify the stroma of the TME which may favor the survival of TICs displaying specific adhesion proteins. We and others have shown that NF-kB transcription factors RelA and RelB respond to signals from the ovarian TME to promote proliferation, chemoresistance, and survival. Our results further show that RelA and RelB regulate expression of specific integrins. Recently we found that TNF-like inducer of apoptosis (TWEAK), a cytokine involved in tissue repair, and its only known receptor Fn14, are enriched following chemotherapy, leading to enhanced chemoresistance and survival of TICs. TWEAK also induces expression of specific integrins in ovarian TICs. Therefore, we hypothesize that NF-kB mediated integrin expression enables adhesion and survival of TICs to a chemotherapy modified extracellular matrix (ECM) to facilitate tumor repopulation and relapse. Using magnetic activated cell sorting (MACS) for CD117+ TICs, we found that a significant majority are enriched for integrin AVB3. Supporting a role for TWEAK-Fn14-NF-kB in mediating integrin expression we further show that integrin AVB3, but not AVB5 or A5B1, was significantly increased in CD117+ TICs in the presence of TWEAK and was dependent on Fn14 as demonstrated through knockdown experiments. RNA-sequencing analysis of tumor cells grown in spheroid conditions with RelA or RelB shRNA knockdown relative to control, show ITGAV is dependent on RelA while ITGB3 expression is dependent on RelB. Taken together, our findings suggest CD117+ TICs have enhanced expression of AVB3 and is further induced by TWEAK mediated NF-kB activity for adaptation to a stressful post-chemotherapy environment. Ongoing in vitro and in vivo studies are investigating the therapeutic benefit of targeting AVB3 or TWEAK in CD117+ TICs for prevention of adhesion and tumor repopulation following chemotherapy. These studies will uncover interactions between TICs and ECM through their unique expression of integrins and clarifies the role of NF-kB transcription factors RelA and RelB in regulating TIC survival in the post-chemotherapy TME. Citation Format: Omar Lujano Olazaba, Mikella Robinson, Cecilia Gallo, Mena Shammas, Katelyn Shelby, Luisjesus Cruz, Samuel F. Gilbert, Carrie D. House. The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4264.

Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.

Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.

Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB–dependent IFITM3 expression

The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape we conducted a gain of function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including JADE3 a protein involved in directing the histone acetyltransferase HBO1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogues JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well characterized antiviral gene Interferon-Induced Transmembrane Protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB dependent IFITM3 expression to restrict influenza A virus infection.

Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

Abstract P34: Bayesian networks modeling identifies a reliance of TP53 mutant AML on NF kappa B signaling

Abstract In acute myeloid leukemia (AML), TP53 mutations ( TP53Mut ) confer the worst prognosis. Leukemia stem cells (LSCs) are rare AML cells endowed with self-renewal capacity, allowing them to recapitulate leukemia after therapy and cause disease progression. In human AML, signaling pathways associated with inflammation have been implicated in the pathogenesis of TP53Mut AML, but the pathways that mediate self-renewal in human TP53Mut AML are not well-known. To define the signaling pathways that are activated in TP53Mut human LSCs, we used CyTOF, a form of flow cytometry that can measure up to 40 proteins simultaneously at single-cell resolution. We compared the levels of activated signaling molecules in the LSC-enriched (CD34+CD38−) subset of seven TP53Mut and seven TP53 wild-type ( TP53WT ) AML samples. Phosphorylated NF kappa B (pNFkB), pSTAT1, and pP38 are significantly higher in TP53Mut AMLs. In contrast, Ki67 and pMAPKAPKII are higher in TP53WT AML. These data demonstrate a unique signaling activation state in TP53Mut AML. The influence of signaling molecules on downstream targets can vary by cellular context. Therefore, we asked whether the influence and interaction of the activated signaling molecules vary between TP53Mut and TP53WT LSCs. To model the signaling architecture, we performed Bayesian networks modeling, a machine learning algorithm that has been validated as a method to discover statistical correlations and dependencies between signaling molecules. We found a similar global signaling network structure and hierarchy in TP53Mut and TP53WT AMLs. However, TP53Mut model showed NFkB have strong influences on phosphorylated Histone H3 (pHIS3) and Ki67, suggesting that proliferation depends on NFkB levels in TP53Mut LSCs. Furthermore, NFkB levels were highly dependent on pSTAT1 in TP53Mut LSCs. Next, we compared the transcriptional profiles of TP53Mut and TP53WT samples in the BEAT AML dataset and found that TP53Mut AML displayed significant enrichment of all the NFkB gene sets (n=19) in the molecular signatures database, providing more evidence for a unique reliance of TP53Mut human AML on NFkB signaling. Therefore, we used lentivirus transduced shRNA constructs to determine the influence of mutant p53 on signaling in human AML. We found that knockdown of TP53Mut reduced NFkB protein levels in Kasumi AML cells (which are TP53R248Q/ − ) but not in MOLM13 AML cells (which are TP53WT). RNA sequencing of transduced Kasumi cells showed that TP53Mut knockdown led to loss of NFkB and LSC self-renewal signatures. Knockdown of TP53Mut in primary human TP53Mut AMLs (S127F/- and S241Y/-) led to a similar loss of NFkB and LSC signatures in these primary samples as well. Colony formation in Kasumi cells and TP53Mut primary human AML were also abrogated after TP53Mut knockdown. These data show TP53Mut promotes NFkB activation in human AML and suggests that human TP53Mut LSCs may be dependent on NFkB for self-renewal. These data implicate NFkB as a possible therapeutic strategy to target TP53Mut human LSCs. Citation Format: Daniel Chang, Klara Noble-Orcutt, Marie Lue Antony, Yoonkyu Lee, Fiona He, Karen Sachs, Chad L Myers, Zohar Sachs. Bayesian networks modeling identifies a reliance of TP53 mutant AML on NF kappa B signaling [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P34.

Abstract A007: Single nuclei analysis of uterine serous carcinoma reveals racial differences in immune signaling

Abstract Endometrial cancer (EC) is a growing public health concern, as incidence rates continue to rise every year. Even more troubling is the unequal survival rates between racial groups with EC, as Black EC patients are dying at twice the rate of white EC patients. Uterine serous carcinoma (USC) is an aggressive subtype of EC that affects Black patients more frequently than white patients. While USC accounts for only 10-15% of all reported EC cases, it has a disease specific mortality rate of over 40%. It is currently unclear what is causing the racial differences in subtype frequency and survival rate of USC. To gain insight on the transcriptomic differences of tumor cells and cells within the tumor microenvironment (TME), we performed single-nuclei RNA sequencing of 13 USC tumors, with 9 tumors from Black patients and 4 from white patients. We found significant gene expression differences, including the upregulation of PAX8 in tumors of Black patients. PAX8 is a lineage factor important in Mullerian duct tissues and is frequently upregulated in EC. In PAX8 high tumor cells, signaling pathways such as TGBF, JAK- STAT, and NFKB activity was enriched, while PAX8 low cells were enriched for p53, WNT, and estrogen signaling. To understand if PAX8 directly affects epithelial immune signaling, we performed RNA-seq on PAX8 knock down (KD) ARK2 cells. Analysis of DEGs showed that PAX8 KD cells express high IL-1A and IL-1B, while chemokines such as CXCL1 and Midkine were downregulated. When conditioned media (CM) from PAX8 KD cells were added to THP-1 macrophages, decreased levels of anti-inflammatory markers such as ARG1 and TGFB1 were observed, compared to PAX8 expressing cells. Additionally, pro-inflammatory molecules such as TNFa and IL-6 were expressed at higher levels. These observations were validated by a cytokine antibody array; macrophages treated with PAX8 KD CM secreted higher levels of pro- inflammatory cytokines such as IL-1A, IL-1B, IL-6, and CXCL5. Anti-inflammatory molecules such as TGFB1 and TGFB2, and growth factors such as FGF6 and FGF7, were secreted at lower levels than control. These studies indicate that PAX8 is a novel regulator of the epithelial- immune axis, which is upregulated in USC compared to normal tissue and modulates the TME in favor of immune suppression and tumor growth. Because PAX8 expression is higher in tumors of Black patients compared to white, this may indicate that Black patients have a higher degree of immunosuppression. Our studies are the first to report tumor differences between Black and white USC patients at the single nuclei level. Additionally, we report findings from the first study to use single nuclei sequencing to study a tumor from Black USC patients. Our results indicate that taking race into account when determining treatment for USC may improve outcomes for Black patients. Citation Format: Grace Foley, J. Julie Kim. Single nuclei analysis of uterine serous carcinoma reveals racial differences in immune signaling [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A007.

Non-coding RNAs and neuroinflammation: implications for neurological disorders.

Neuroinflammation is considered a balanced inflammatory response important in the intrinsic repair process after injury or infection. Under chronic states of disease, injury, or infection, persistent neuroinflammation results in a heightened presence of cytokines, chemokines, and reactive oxygen species that result in tissue damage. In the CNS, the surrounding microglia normally contain macrophages and other innate immune cells that perform active immune surveillance. The resulting cytokines produced by these macrophages affect the growth, development, and responsiveness of the microglia present in both white and gray matter regions of the CNS. Controlling the levels of these cytokines ultimately improves neurocognitive function and results in the repair of lesions associated with neurologic disease. MicroRNAs (miRNAs) are master regulators of the genome and subsequently control the activity of inflammatory responses crucial in sustaining a robust and acute immunological response towards an acute infection while dampening pathways that result in heightened levels of cytokines and chemokines associated with chronic neuroinflammation. Numerous reports have directly implicated miRNAs in controlling the abundance and activity of interleukins, TGF-B, NF-kB, and toll-like receptor-signaling intrinsically linked with the development of neurological disorders such as Parkinson's, ALS, epilepsy, Alzheimer's, and neuromuscular degeneration. This review is focused on discussing the role miRNAs play in regulating or initiating these chronic neurological states, many of which maintain the level and/or activity of neuron-specific secondary messengers. Dysregulated miRNAs present in the microglia, astrocytes, oligodendrocytes, and epididymal cells, contribute to an overall glial-specific inflammatory niche that impacts the activity of neuronal conductivity, signaling action potentials, neurotransmitter robustness, neuron-neuron specific communication, and neuron-muscular connections. Understanding which miRNAs regulate microglial activation is a crucial step forward in developing non-coding RNA-based therapeutics to treat and potentially correct the behavioral and cognitive deficits typically found in patients suffering from chronic neuroinflammation.

TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

A multi-organ, feto-maternal interface organ-on-chip, models pregnancy pathology and is a useful preclinical extracellular vesicle drug trial platform

Pregnant women and their fetuses are often excluded from clinical trials due to missing drug-related pre-clinical trial information at the human feto-maternal interface (FMi). The two interfaces-placenta/decidua and fetal membranes/decidua are gatekeepers of drug transport; however, testing their functions is impractical during pregnancy. Limitations of current in-vivo/in-vitro models have hampered drug development and testing during pregnancy. Hence, major complications like preterm births and maternal and neonatal mortalities remain high. Advancements in organ-on-chip (OOC) platforms to test drug kinetics and efficacy and novel extracellular vesicle-based fetal drug delivery are expected to accelerate preclinical trials related to pregnancy complications. Here we report the development and testing of a humanized multi-organ fetal membrane/placenta (fetal)-decidua (maternal) interface OOC (FMi-PLA-OOC) that contains seven cell types interconnected through microchannels to maintain intercellular interactions as seen in-utero. Cytotoxicity, propagation, mechanism of action, and efficacy of engineered extracellular vesicles containing anti-inflammatory interleukin (IL)-10 (eIL-10) were evaluated to reduce FMi inflammation associated with preterm birth. A healthy and disease model (lipopolysaccharide-infectious inflammation) of the FMi-PLA-OOC was created and co-treated with eIL-10. eIL-10 propagated from the maternal to fetal side within 72-h, localized in all cell types, showed no cytotoxicity, activated IL-10 signaling pathways, and reduced lipopolysaccharide-induced inflammation (minimized NF-kB activation and proinflammatory cytokine production). These data recapitulated eIL-10s’ ability to reduce inflammation and delay infection-associated preterm birth in mouse models, suggesting FMi-PLA-OOC as an alternative approach to using animal models. Additionally, we report the utility of eIL-10 that can traverse through FMis to reduce inflammation-associated pregnancy complications.

Pulsed Hyperoxia Acts on Plasmatic Advanced Glycation End Products and Advanced Oxidation Protein Products and Modulates Mitochondrial Biogenesis in Human Peripheral Blood Mononuclear Cells: A Pilot Study on the "Normobaric Oxygen Paradox".

The "normobaric oxygen paradox" (NOP) describes the response to the return to normoxia after a hyperoxic event, sensed by tissues as an oxygen shortage, up-regulating redox-sensitive transcription factors. We have previously characterized the time trend of oxygen-sensitive transcription factors in human PBMCs, in which the return to normoxia after 30% oxygen is sensed as a hypoxic trigger, characterized by hypoxia-induced factor (HIF-1) activation. On the contrary, 100% and 140% oxygen induce a shift toward an oxidative stress response, characterized by NRF2 and NF-kB activation in the first 24 h post exposure. Herein, we investigate whether this paradigm triggers Advanced Glycation End products (AGEs) and Advanced Oxidation Protein Products (AOPPs) as circulating biomarkers of oxidative stress. Secondly, we studied if mitochondrial biogenesis was involved to link the cellular response to oxidative stress in human PBMCs. Our results show that AGEs and AOPPs increase in a different manner according to oxygen dose. Mitochondrial levels of peroxiredoxin (PRX3) supported the cellular response to oxidative stress and increased at 24 h after mild hyperoxia, MH (30% O2), and high hyperoxia, HH (100% O2), while during very high hyperoxia, VHH (140% O2), the activation was significantly high only at 3 h after oxygen exposure. Mitochondrial biogenesis was activated through nuclear translocation of PGC-1α in all the experimental conditions. However, the consequent release of nuclear Mitochondrial Transcription Factor A (TFAM) was observed only after MH exposure. Conversely, HH and VHH are associated with a progressive loss of NOP response in the ability to induce TFAM expression despite a nuclear translocation of PGC-1α also occurring in these conditions. This study confirms that pulsed high oxygen treatment elicits specific cellular responses, according to its partial pressure and time of administration, and further emphasizes the importance of targeting the use of oxygen to activate specific effects on the whole organism.

Quercetin inhibits NF-kB and JAK/STAT signaling via modulating TLR in thymocytes and splenocytes during MSG-induced immunotoxicity: an in vitro approach.

The most widely used food additive monosodium glutamate (MSG) has been linked to immunopathology. Conversely, quercetin (Q), a naturally occurring flavonoid has been demonstrated to have immunomodulatory functions. Therefore, the purpose of the study is to determine if quercetin can mitigate the deleterious effects of MSG on immune cells, and the possible involvement of TLR, if any. METHODS AND RESULTS: This study was conducted on Q, to determine how it affects the inflammatory response triggered by MSG in primary cultured thymocytes and splenocytes from rats (n = 5). Q shielded cells by augmenting cell survival and decreasing lactate dehydrogenase leakage during MSG treatment. It decreased IL-1β, IL-6, IL-8, and TNF-α expression and release by hindering NF-kB activation and by inhibiting the JAK/STAT pathway. Moreover, Q prevented NLRP3 activation, lowered IL-1β production, and promoted an anti-inflammatory response by increasing IL-10 production. Q reduced MSG-induced cellular stress and inflammation by acting as an agonist for PPAR-γ and LXRα, preventing NF-kB activation, and lowering MMP-9 production via increasing TIMP-1. Additionally, Q neutralized free radicals, elevated intracellular antioxidants, and impeded RIPK3, which is involved in inflammation induced by oxidative stress, TNF-α, and TLR agonists in MSG-treated cells. Furthermore, it also modulated TYK2 and the JAK/STAT pathway, which exhibited an anti-inflammatory effect. MSG exposure is associated with immune cell dysfunction, inflammation, and oxidative stress, and Q modulates TLR to inhibit NF-kB and JAK/STAT pathways, providing therapeutic potential. Further research is warranted to understand Q's downstream effects and explore its potential clinical applications in inflammation.