NF-kappaB Activation Research Articles

Abstract 385: Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma

Abstract Background: IRAK4, a serine/threonine kinase, was identified as a therapeutic target in hematological malignancies. IRAK4-mediated activation of NF-kappaB could play an important role in NF-kappaB-regulated survival and chemoresistance of cancer cells. CA-4948, a first-in-class small molecule inhibitor of IRAK4, has been tested in Phase 1 clinical trial with evidence of clinical activity in Non-Hodgkin's Lymphoma (NHL) patients. To support the development of companion diagnostic for CA-4948, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers of response to CA-4948 in tumor biopsy samples obtained from NHL patients. Materials and Methods: IHC assay was developed and used for analysis of NF-kappaB p-p50 and p-IRAK1 expression in tumor biopsy samples obtained from 14 patients with NHL. Patients were defined according to their clinical response to CA-4948 treatment: stable disease (SD), 6 cases and progressive disease (PD), 8 cases. Results: We found nuclear and/or cytoplasmic expression of NF-kappaB p-p50 in all 6 SD cases treated with 50 mg QD (2 cases, tumor regression), 50 mg BID (1 case), 200 mg BID (1 case, tumor regression) and 400 mg BID (2 cases). Expression of NF-kappaB p-p50 was not detected in 7 of 8 cases with PD including patients treated with 50 mg QD (1 case), 100 mg QD (1 case), 100 mg BID (3 cases), 200 mg BID (1 case) and 400 mg BID (1 case). We found statistically significant correlation between expression of NF-kappaB p-p50 in tumor biopsy and SD in NHL patients treated with CA-4948 (p<0.05). Analysis of NF-kappaB p-p50 expression in paired tumor biopsy samples (3 cases) collected before and after the treatment with CA-4948 revealed a significant downregulation of NF-kappaB p-p50 expression in tumors obtained from CA-4948-treated NHL patients. In support of our in vivo findings, our in vitro experiments demonstrated that expression of NF-kappaB p-p50 was depleted in 3D lymphoma organoids treated with a clinically relevant concentration of CA-4948. Our results support further development of NF-kappaB p-p50 as a potential predictive and pharmacodynamic biomarker of IRAK4 inhibitor CA-4948. We found that expression of p-IRAK1 in tumor biopsies was not correlated with clinical response. Conclusions: Although the cohort size is small, our findings suggest that expression of NF-kappaB p-p50 can serve as biomarker to predict SD in response to the treatment with IRAK4 inhibitor CA-4948 in NHL patients. NF-kappaB p-p50 selection strategy might be used in future clinical trials to identify NHL patients which are most likely to respond to CA-4948 in combination with chemotherapy or targeted therapeutics. Citation Format: Andrey Ugolkov, Maria Samson, Rosanna Hok, Reinhard von Roemeling, Robert Martell. Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 385.

Mesenchymal Stromal Cell Sialylation Enhances Immune Suppression in Multiple Myeloma

Introduction: In multiple myeloma (MM) the bone marrow (BM) microenvironment, plays an important role in mediating survival, proliferation, resistance to therapy as well as disease evolution. The BM microenvironment is mainly composed of mesenchymal stromal cells (BM-MSC), endothelial cells, immune cells, and extracellular matrix. BM-MSC can acquire distinct functional phenotypes that are influenced by signals in the microenvironment and have been shown to facilitate MM development and drug resistance through cell-cell contact and cytokine secretion. However, little is known about the mechanisms of immune modulation mediated by BM-MSC in the inflammatory MM tumour microenvironment (TME). Aberrant glycosylation is a hallmark of cancer cells, playing an important role in tumor progression. Our data strongly links aberrant sialylation with poor prognosis in MM (1) but to date the immunological consequences of this phenomenon have not been explored. Here we sought to investigate if regulation of BM-MSC sialylation alters their ability to inhibit effector T-cell function in an inflammatory microenvironment, characteristic of the MM TME.Methods: BM-MSC were isolated from BALB/c mice and extensively characterized in vitro . Since inflammatory mediators, such as TNF-α and IL-1β are present at high levels in MM due to constitutive NF-kappaB activity, we assessed the effects of both of these pro-inflammatory mediators on BM-MSC phenotype. BM-MSC were treated with both tumor necrosis factor alpha (TNF-α 50ng/ml) and interleukin 1 beta (IL-1β 50ng/ml) (iMSC) for 72 hours and cell surface sialylation was analysed by flow cytometry. To determine immunoregulatory ability, BM-MSC and iMSC were co-cultured in mixed lymphocyte reactions (MLRs) for 96 hours. T-cell proliferation, activation, death and differentiation were determined by flow cytometry. To determine the role of increased sialylation in lymphocyte suppression, both BM-MSC and iMSC were pre-treated with a sialyltransferase inhibitor (3Fax-Neu5Ac) for 72 hours prior to TNF-α and IL-1β stimulation. To determine the important of antigen presenting cells in sialic acid dependent regulation of T cell phenotype, CD11c+ and CD11b+ innate immune cells were removed via MACS sorting prior to co-culture. BM-MSC: Lymphocyte co-culture supernatants for nitric oxide (NO), IL-10 and PGE-2 were analysed by ELISA.Results: iMSC displayed significant phenotypical changes with CD73 and CD44 increasing significantly. Both SCA-1 and CD105 were significantly reduced. No changes in cell viability were noted after pre-treatment and no significant changes in proliferation were observed. iMSC had significantly increased levels of both α2-3 and α2-6 linked sialic acid when compared to BM-MSC. In vitro, iMSC displayed an enhanced ability to inhibit lymphocyte proliferation when compared to BM-MSC alone. iMSC significantly inhibited the proliferation of hyper-stimulated CD3+CD4+ and CD3+CD8+ lymphocytes. 3Fax-Neu5Ac treated BM-MSC and iMSC displayed no significant changes in phenotype, viability, proliferation and cell size. However, following sialyltransferase inhibition, iMSC lost the ability to suppress both CD3+CD4+ and CD3+CD8+ lymphocytes resulting in significant restoration of lymphocyte proliferation. This restoration of proliferation was further enhanced in the absence of CD11c+ and CD11b+ innate immune cells. Cell surface sialic acid expression correlated with both CD3+CD4+ and CD3+CD8+ lymphocyte suppression. No changes in soluble mediators of BM-MSC immunomodulation, such as nitic oxide, IL-10 and PGE-2 were observed suggesting that restoration in proliferation was both sialic acid and cell-contact dependent.Conclusions: Our findings confirm inflammation, characteristic of the MM TME, induces BM-MSC sialylation and enhances their ability to suppress activated adaptive and innate immune effectors. Understanding the potential utility of targeting BM-MSC sialylation and consequently their immunomodulatory potential, may enhance immune cell activation in the MM microenvironment, providing further rationale to targeting BM-MSC sialylation in the context of MM. We suggest that understanding the functional importance of the BM stroma and its interaction with MM and immune cells is likely to lead to the identification of sialylation, as a new molecular target. 1.Glavey SV, et al Blood. 2014 Sep 11;124(11):1765-76 [Display omitted] DisclosuresRyan:Janssen: Research Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Mono-macrophage-Derived MANF Alleviates Bacterial Myocarditis by Inhibiting NF-kappaB Activation and Myocardial Inflammation.

Bacterial myocarditis is a key cause leading to myocardial damage and cardiac dysfunction. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been found to be an anti-inflammatory factor. This study is to explore the effect of MANF on LPS-induced myocardial inflammation and macrophage differentiation. The myocarditis mouse model was constructed by LPS treatment. Myocardial damage and serum inflammatory factors were evaluated by ELISA. RT-qPCR was used to detect mRNA of M1/M2 macrophage markers. Western blot, immunohistochemical, and immunofluorescent staining were used to examine myocardial M1/M2 macrophages and NF-κB activation. Mono-macrophage-derived MANF deficiency enhanced LPS-induced inflammatory response and increased M1 macrophages in myocardium tissues, further causing more severe myocardial injury and lower survival rate of mice. Also, LPS-induced myocardial NF-κB activation was strengthened after mono-macrophage-derived MANF knockout. Mono-macrophage-derived MANF inhibits bacterial myocarditis and myocardial M1 macrophage differentiation, which is potential to be used for bacterial myocarditis treatment clinically.

Proximity Dependent Labeling Describes Role of Abelson Interactor 1 in Centrosome Biology

Abelson interactor 1 (Abi-1) is a signaling adaptor protein dysregulated in cancer. Abi-1 is established to be involved in cytoskeletal reorganization regulated by Ras to Rac GTPase signal transduction, and our group recently showed that murine Abi-1 deficiency causes myeloproliferative neoplasm-like phenotype linked to activation of Src family kinases, STAT3, and NF-kappaB (Chorzalska et al, Blood, 2018). To resolve molecular mechanisms by which Abi-1 dysregulation destabilizes inflammatory homeostasis leading to cancer, we used proximity dependent biotin labeling followed by mass spectrometry (MS) using TurboID linked to Abi-1. TurboID is a biotin ligase from E. coli catalytically enhanced so that it rapidly converts biotin to active biotinoyl-5’-AMP which then binds to local proteins, enabling enrichment of biotinylated proteins from lysates by streptavidin and identification by MS (Brannon, Nat Biotech, 2018) (A). We used a retroviral transduction system to generate stable mouse embryonic fibroblast (MEF, NIH/3T3) cell lines expressing TurboID or TurboID-Linker-Abi-1 (B). We performed three experiments of labeling followed by MS using TurboID control, TurboID-Abi-1, and wildtype cell lines. Label-free quantification MS and data processing was performed using a Q Exactive Plus LC-MS/MS and Proteome Discoverer 2.3. Probable Abi-1 interacting proteins were determined by comparing assigned protein peak areas (PA) and peptide spectral matches (PSM) between streptavidin-enriched lysates from MEFs expressing TurboID-Abi-1 vs. TurboID, and TurboID-Abi-1 vs. wildtype (C). Valid statistical thresholds were determined by retrospective analysis of known interactor enrichment assisted by in-house software “Interactomatiker”. We identified 4,082 proteins, 2,997 in TurboID-Abi-1 lysates. Retrospective analysis of MS data determined a statistical threshold defining most probable Abi-1 interactors as: TurboID-Abi-1 vs. controls PA ratio ≥ 1.5, FDR ≤ 0.05, and PSM ratio ≥ 3.5, FDR ≤ 0.001, with a minimum average of 1 PSM in TurboID-Abi-1 MS injections. 74 proteins met this statistical threshold, 20 of which are known Abi-1 interactors (D). Pathway analyses of most probable interactors identified expected Abi-1 role in cytoskeletal reorganization (E), but also indicate a new role in centrosome biology by interaction with Nde1, Haus5, Haus7, Cep131, and Pcm1 (F). Additionally, Abi-1 deficient or over-expressing cells show increased or decreased proliferation by EdU incorporation assay (G). TurboID using Abi-1, assessed by expected enrichments, identified new Abi-1 roles which may provide mechanistic context to it's role in cancer. In addition to affecting centrosome biology, less stringent filtering strategy also indicates possible roles in TNFR signaling and protein trafficking. Using a discovery-based method to assess adaptor protein interactions, we identified signaling nodes likely affected in cancers associated with dysregulated Abi-1 activity.

NOVEL PATHOPHYSIOLOGICAL ROLES OF ALPHA-SYNUCLEIN (SNCA) IN AGE-RELATED VASCULAR ENDOTHELIAL DYSFUNCTION

Objective: Although SNCA is one of the well-known pathological molecule of synucleinopathy in neurons, its physiological roles remain to be identified. In the present study, we sought to elucidate the novel functions of SNCA in endothelium. Design and method: Analysis for human samples, in vitro experiments using endothelial cells (ECs), and in vivo investigation using SNCA knockout or SNCA/ApoE double knockout mice were performed. Results: We found SNCA was expressed in and secreted from ECs by functional screening for anti-inflammatory molecules and detectable in circulating blood. Intriguingly, the human population study revealed serum SNCA levels decreased with aging and displayed inverse correlation with blood pressure and insulin sensitivity, which indicated protective properties of circulating SNCA on vascular endothelial function. Furthermore, we also found SNCA knockout mice displayed phenotypes of metabolic syndrome such as hypertension, impaired glucose metabolism, and dyslipidemia. Based on these preliminary data, we sought to elucidate the physiological functions of endogenous and exogenous SNCA for ECs. Exogenous treatment with recombinant SNCA (rSNCA) promoted eNOS activation and nitric oxide production via Gab1/PI3K/Akt pathway in ECs, followed by cGMP production in co-cultured vascular smooth muscle cells. Treatment with rSNCA also suppressed TNF-alpha induced NF-kappaB activation in ECs. As to endogenous SNCA, replicative senescence showed attenuation of SNCA expression in ECs and siRNA-mediated silencing of SNCA induced eNOS inactivation and cell senescence assessed by beta-gal activity along with decreased Sirt1 expression and increased p53 expression. SNCA overexpression displayed NF-kappaB inactivation in ECs. In ex vivo study, aortas from SNCA knockout mice showed impairment of acetylcholine-induced relaxation possibly due to eNOS dysfunction. In in vivo study utilizing atherosclerosis model of SNCA/ApoE double-knockout mice showed exaggerated expression of inflammatory genes which play important roles in atherogenesis. Conclusions: In conclusion, these results indicate exogenous and endogenous SNCA in ECs might physiologically maintain cerebral vascular integrity. Aging or aggregation-related loss-of-function of SNCA in ECs might be partially correlated with age-related hypertension or clinical features of dementia with Lewy body such as fluctuating cognitive function and marked sensitivity of antipsychotic which could be modified by cerebral blood flow and vascular permeability.

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Mechanisms of Adiponectin in Regulation of Proinflammatory Cytokine Production and Migration in Macrophages.

IntroductionEvidence indicate that adiponectin may exert pro-inflammatory effects on inflammatory cells. We have found that adiponectin knockout decreased inflammatory reaction and tubular damage in the ischemia-reperfusion kidney in APN-knockout mice. Globular adiponectin and full-length adiponectin (g-APN and f-APN) were used in this study to investigate the effects of adiponectin on proinflammatory cytokines production and migration in Raw 264.7 cells.MethodsProinflammatory cytokines production was detected by real-time RT-PCR. NF-kappaB activation was interrupted through Ad-DN-IκBα or SN-50 to confirm how g-APN induces proinflammatory cytokines production. The siRNA against AdipoR1 and AdipoR2 was investigated to uncover the signaling pathway that may involve in NF-kappaB activation and migration in Raw 264.7 cells.Resultsg-APN, not f-APN, was found triggering the production of inflammatory cytokine MIP-2, IL-6, TNFα, and MCP-1 in Raw 264.7 cells. NF-kappaB Inhibition by Ad-DN-IκBα expression or cell-permeable NF-κB inhibitor SN-50 could decrease NF-kappaB nuclear translocation and subsequently decrease inflammatory cytokine expression triggered by globular ANP. However, AdipoR1 and AdipoR2 were not found involved in NF-kappaB activation in this study. Full-length APN, not g-APN, was involved in the promotion of macrophage migration. The migration induced by full-length APN could be inhibited by knockdown of AdipoR1 expression with siRNA. The migration effect could also be inhibited by PI3Kγ inhibitor, AS-605240.DiscussionThese results suggested that full-length adiponectin increases macrophage migration through Adiponectin-AdipoR1-PI3Kgamma signaling pathway. However, NF-κB activation induced by g-APN in this study was independent of AdipoR1 or AdipoR2. The exact signaling pathway of NF-κB activation by adiponectin should be further studied to find a new anti-inflammatory target.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Аdvanced glycation end-products as novel biomarkers of eosinophil-derived lung inflammatory diseases (literature review)

Advanced glycation end-products (AGEs) are created during the process of glycation of cells from various tissues and fluids and are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the amino group of proteins, lipids, and nucleic acid. In normal conditions, they play the immunoregulatory role. In pathologic conditions AGEs activate the receptors for advanced glycation end products (RAGE) and cause long-lasting inflammation. RAGE participates actively in various disorders such as rheumatoid arthritis, diabetes, etc. However, there is relatively small number of scientific studies on the possibility of using the role of AGE in the pathogenesis of allergic diseases. RAGE transcript and protein are expressed in the lung by pulmonary type I alveolar epithelial cells, suggesting that RAGE has an important role in lung pathophysiology. They repress some endogenous autoregulatory functions leading to many diseases, including allergy. Oxidative stress increases the inflammatory reaction in asthma and allergies. Long-lasting inflammation followed by free radicals production are important factors involved in allergic reactions, they negatively influence the incidence and prognosis of allergy. RAGEs are expressed on circulating immune cells, they activate NF kappaB and intracellular oxidative stress also increases the inflammatory reaction in asthma and allergies. The membrane RAGE (mRAGE) signaling is proinflammatory, whereas soluble RAGE (sRAGE), a secreted form of RAGE, is generally anti-inflammatory. The study of AGEs, soluble RAGE, ligands of RAGE HMGB1, and S100A8/A913 and IL-33 is useful in the context of their considering as biomarkers to the differentiation diagnostic between eosinophils-derived and neutrophil-derived asthma/AAD. The mean serum levels of RAGE may be the target of new therapeutic interventions.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

ReactomeGSA - Efficient Multi-Omics Comparative Pathway Analysis

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

Abstract IA49: MALT1 targeting for B-cell lymphomas

Abstract IA49: MALT1 targeting for B-cell lymphomas

PA-X protein of H5N1 avian influenza virus inhibits NF-kappaB activity, a potential mechanism for PA-X counteracting the host innate immune responses

PA-X is a fusion protein of influenza virus which plays a crucial role in modulating influenza virus-induced host innate immune response and subsequent pathogenicity. However, the potential mechanism of PA-X regulation of the host innate immune response remains largely unknown. It is well known that NF-κB signal pathway is crucial for the immediate early step of immune responses activation, while the specific role of PA-X in NF-κB transcriptional activity is totally unknown. In this study, we initially showed that PA-X inhibits NF-κB transcription that stimulated by poly(I:C). We then further determined that the inhibitory effect on NF-κB activation mediated by PA-X was characterized by restricting NF-κB p65 nuclear translocation and nuclear NF-κB p65 activity but not by impeding the phosphorylation of NF-κB p65. Correspondingly, PA-X decreases the amount of NF-κB signaling pathway-associated genes, including TNF-α, Nos2, IL-6 and IL-2. Moreover, PA-X also suppresses both the mRNA and protein expression level of IFN-β, suggesting the direct contribution of PA-X to the inhibition of NF-κB-regulated IFN-β expression. Together, our study sheds light on the potential molecular mechanisms underlying the regulation of host NF-κB activity by PA-X and also identifies a novel functional role for PA-X in counteracting the host innate immune response. However, further exploration of the more elaborate mechanism of PA-X-mediated inhibition of NF-κB activity and the associated signaling pathway may help to elucidate its precise mechanism of evading and subverting the host immune response.

Understanding Selenium and Glutathione as Antiviral Factors in COVID-19: Does the Viral Mpro Protease Target Host Selenoproteins and Glutathione Synthesis?

Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally link selenium and glutathione, which both show correlations with clinical outcomes in COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact with an inactive C145A mutant of Mpro, the main cysteine protease of SARS-CoV-2, but not with catalytically active wild-type Mpro. This seemingly anomalous result is what might be expected if GPX1 is a substrate for the active protease, leading to its fragmentation. We show that the GPX1 active site sequence is substantially similar to a known Mpro cleavage site, and is identified as a potential cysteine protease site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent with previously documented effects of recombinant SARS-CoV Mpro in transfected cells, including increased reactive oxygen species and NF-κB activation. Because NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could contribute to increased inflammation and cytokine storms observed in COVID-19. Using web-based protease cleavage site prediction tools, we show that Mpro may be targeting not only GPX1, but several other selenoproteins including SELENOF and thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme for glutathione synthesis. This hypothesized proteolytic knockdown of components of both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production. The resulting “collateral damage” of increased oxidative stress and inflammation would be exacerbated by dietary deficiencies of selenium and glutathione precursors.

Estrogen receptor regulates immune defense by suppressing NF-κB signaling in the Crassostrea hongkongensis

The crosstalk between the estrogen receptor (ER) and NF-κB signalling pathways has merged in vertebrates and plays a key role in the control of genes involved in inflammation, cell proliferation and apoptosis. However, such crosstalk between the endocrine and immune systems needs to be explored in lower invertebrates. In this study, we identified a 2856-bp homologue of the estrogen receptor from Hong Kong oyster (ChER), containing a 5′ untranslated region (UTR) of 234 bp, a 3’ UTR of 387 bp, and an open reading frame (ORF) of 2235 bp. We observed that overexpression of ChER suppressed ChRel-dependent NF-kappaB (NF-κB) activation in the HEK293T (human embryonic kidney 293T) cell line, and depletion of ChER in vivo resulted in upregulation of two NF-κB-responsive marker genes, namely, TNF-α and IL-17, which confirmed its potential role in controlling NF-κB signalling. Furthermore, an EMSA (electrophoretic mobility shift assay) showed that ChER could negatively regulate the binding of ChRel to NF-κB probe-responsive elements. Serial domain requirement analysis showed that both region C (DNA-binding domain) and region E (ligand-binding domain) of ChER were essential for mediating the crosstalk underlying ChER-dependent NF-κB suppression. In conclusion, we demonstrate for the first time the negative regulatory role of the ER in NF-κB signalling in oysters, strongly indicating the presence of complex crosstalk between the endocrine and immune systems in lower marine molluscs.

Nucleolin Mediates LPS-induced Expression of Inflammatory Mediators and Activation of Signaling Pathways

SummaryIn this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1β and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1β and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1β and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1).

Dynamic polarization shifting from M1 to M2 macrophages in reduced osteonecrosis of the jaw-like lesions by cessation of anti-RANKL antibody in mice.

Denosumab-related osteonecrosis of the jaw (DRONJ), which mainly occurs in cancer patients receiving anti-receptor activator NF-kappaB ligand (RANKL) antibody, reduces oral health-related quality of life. However, the exact mechanisms of and definitive treatment strategies for DRONJ remain unknown. We hypothesized that cessation of denosumab heals and/or ameliorates DRONJ, since it is a protein-based antibody agent, although stopping denosumab should be avoided in clinical situations. Therefore, the aims of this study were: 1) to create a healing and/or amelioration murine model of DRONJ-like lesions induced by chemotherapy/anti-RANKL antibody (mAb) combination therapy and tooth extraction; and 2) to investigate histopathology and immunopathology in the extraction sockets by comparing the murine model of DRONJ-like lesions with the amelioration/healing model of DRONJ-like lesions. Eight-week-old, female C57B/6J mice received chemotherapeutic drug (cyclophosphamide: CY) and mAb combination therapy (CY/mAb) with tooth extraction. Open wounds were sustained in CY/mAb-treated mice at 2 and 4 weeks post-extraction. Impaired socket healing was diagnosed as CY/mAb-related ONJ-like lesions at 3 weeks post-extraction in this study. Next, mAb was discontinued for 2 and 4 weeks after diagnosis of CY/mAb-related ONJ-like lesions. mAb cessation for 2 weeks induced partial osseous wound healing and significantly improved soft tissue wound healing of the extraction sockets. Anti-angiogenesis and normal lymphangiogenesis with CY/mAb combination therapy was not changed by mAb discontinuation. However, mAb cessation for 2 weeks significantly increased the number of CD38+F4/80+ M1 and CD163+F4/80+ M2 macrophages, which significantly increased the M2/M1 ratio in the connective tissue of extraction sockets. No direct effects of mAb on macrophages were noted both in vivo and in vitro. Therefore, the developed healing and/or amelioration murine model of CY/mAb-related ONJ-like lesions is a useful tool to investigate the histopathology and immunopathology of DRONJ in humans. Dynamic polarization shifting from M1 to M2 macrophages induced by mAb cessation may play an important role in wound healing, rather than angiogenesis and lymphangiogenesis, in DRONJ.

Cell-Type Targeted NF-kappaB Inhibition for the Treatment of Inflammatory Diseases.

Deregulated NF-k activation is not only involved in cancer but also contributes to the pathogenesis of chronic inflammatory diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). Ideally, therapeutic NF-KappaB inhibition should only take place in those cell types that are involved in disease pathogenesis to maintain physiological cell functions in all other cells. In contrast, unselective NF-kappaB inhibition in all cells results in multiple adverse effects, a major hindrance in drug development. Hitherto, various substances exist to inhibit different steps of NF-kappaB signaling. However, powerful tools for cell-type specific NF-kappaB inhibition are not yet established. Here, we review the role of NF-kappaB in inflammatory diseases, current strategies for drug delivery and NF-kappaB inhibition and point out the “sneaking ligand” approach. Sneaking ligand fusion proteins (SLFPs) are recombinant proteins with modular architecture consisting of three domains. The prototype SLC1 binds specifically to the activated endothelium and blocks canonical NF-kappaB activation. In vivo, SLC1 attenuated clinical and histological signs of experimental arthritides. The SLFP architecture allows an easy exchange of binding and effector domains and represents an attractive approach to study disease-relevant biological targets in a broad range of diseases. In vivo, SLFP treatment might increase therapeutic efficacy while minimizing adverse effects.

The Anti-inflammatory Effect of Honeysuckle Flowers (Lonicera japonica Thunb., Caprifoliaceae) in Combination with Oroxylum Indicum (Oroxylum indicum Vent. Bignoniaceae)


 This study investigates the anti-inflammatory effect of the honeysuckle flower in combination with Oroxylum indicum in search of a new kind of anti-inflammatory therapy drugs with stronger anti-inflammatory effects and without unwanted adverse reactions. The study results show that a honeysuckle flower dose of 0.72 g/kg/day, Oroxylum indicum dose of 1.2 g/kg/day together with honeysuckle flower combined with Oroxylum indicum with 1:1 ratio had an acute anti-inflammatory effect on the white rat on a mouse Leg Edema Model with carrageenin and the model causing mouse peritonitis. The results also show that a honeysuckle flower dose of 1.44 g/kg/day, Oroxylum indicum the dose of 2.4 g/kg/day together with honeysuckle flower combined with Oroxylum indicum with 1:1 ratio had a chronic anti-inflammation effect on the white mouse on the model causing chronic inflammation of the granuloma. These results open up a subsequent research direction on the possibility of combining various anti-inflammatory medicinal herbs to obtain better anti-inflammatory efficacy and contribute to the promoting of the effect of the available domestic medicinal resources.
 Keywords
 Honeysuckle flowers, Oroxylum indicum, anti-inflammatory effect
 References
 [1] Vu Trieu An, Immunology, Hanoi Medical Publishing House, 2001, pp. 250 - 252.[2] Nguyen Ngoc Lanh, Pathophysiology, Medical Publishing House, 2012, pp. 209 - 229.[3] Le Dinh Roanh, Nguyen Dinh Mao, Inflammatory and infectious pathology, Medical Publishing House,1997, pp. 1 - 132.[4] Vo Van Chi, Dictionary of Vietnamese medicinal plants, Medicine Publishing House,1997, pp. 633-634.[5] Paulina Melega, Lonicera japonica Flower, Veterinarian Reviewed on June 20 (2012).[6] Jin Tae, Seung-Woo Han, et al., Anti-inflammatory effect of Lonicera japonica in proteinase-activated receptor 2-mediated paw edema, Clinica Chimica Acta, Apr; 330(1-2) (2003) 165 - 171.[7] Wie Jong Kwar, Chang Kyun Han, et al., Loniceroside C, an Anti-inflammatory Saponin from Lonicera japonica, Chem. Pharm. Bull (Tokyo) Mar; 51(3) (2003) 333-335.[8] JH. Lee, WS. Ko, YH. Kim, et al., Anti-inflammatory effect of the aqueous extract from Lonicera japonica flower is related to inhibition of NF-KappaB activation through reducing I-kappaBalpha degradation in rat liver, Int J Mol Med, Jan; 7(1) (2001) 79 - 83.[9] Min Ho Han, Won Sup Lee, Arulkumar Nagappan, et al., Flavonoid Isolated from Flowers of Lonicera japonica Thunb. Inhibit Inflammatory Responses in BV2 Microglial Cells by Suppressing TNF-α and IL-β Through PI3K/Akt/NF-Kb Signaling Pathways, Phytotherapy Research, 30(11) (2016) 1824 - 1832. [10] Seung-Hwan Kwon, Shi-Xun Ma, Sa-lk Hong, et al., Lonicera japonica Thunb. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells, J Med Food, Jul 1. 18(7) (2015) 762-775.[11] Do Tat Loi, Vietnamese medicinal plants and herbs, Thoi Dai Publishing House, (2011), 75 - 77, 726 - 728.[12] Mamatha Karnati, Rodda H Chandra, Ciddi Veeresham, et al., Anti-arthritic activity of root bark of Oroxylum indicum (L.) vent against adjuvant-induced arthritis, Pharmacognosy Research, Apr-Jun 5(2) (2013) 121-128.[13] Vietnam Pharmacopoeia Council, Vietnam Pharmacopoeia IV, Medical Publishing House, 2009, pp. 806- 856.[14] C.A. Winter, E.A Risley and G.W Nuss, Carrageenin induced edema in hind paw of the rat as an assay for anti-inflammatory drugs, Proc. Soe. Exp. Bio. Med, No 111 (1962) 544 - 547.[15] Julon, J.C.Guyonnet, et al., Etude des propriétés pharmacologiques d’un nowel anti-inflammatoire, J.Pharmacol (Paris), 3(2) (1971) 259 - 286.

Therapeutic effects of KANK2 in myocardial infarction rats might be associated with the NF-κB p65 inhibition

ObjectiveKN motif and ankyrin repeat domains 2 (KANK2) may inhibit the activation of (NF-kappaB) p65, which plays a role in myocardial injury. Thus, our study aims to discover the effect of KANK2 on myocardial infarction (MI) induced by ligating the left anterior descending coronary artery (LAD) through regulating NF-κB p65 in vivo. MethodsMI rats underwent LAD ligation were administered with intramyocardial injections of KANK2/Control activation plasmids. Six weeks after MI, pressure-volume (P/V) loops was used to investigate the cardiac function of rats, then the following detections were performed, including TTC staining, HE staining, immunofluorescence, Masson’ s trichrome staining, ELISA assay, TUNEL staining, immunohistochemistry, qRT-PCR and Western blotting. ResultsMI rats decreased in maximum pressure (pmax), ejection fraction (EF%), peak rate of pressure rise (dpdtmax) and decline (-dpdtmax) with increased end diastolic pressure (EDP), which was partially reversed by KANK2 overexpression. Besides, KANK2 CRISPR activation plasmids reduced infarct size with less collagen fiber proliferation and neutrophil infiltration in infarct tissues, as well as suppressed pro-inflammatory factors expressions in MI rats. Moreover, injection of KANK2 activation plasmid decreased collagen deposition, aggravated cardiomyocyte apoptosis, enhanced the capillary density, and increased the expressions of VEGF and bFGF in the infarct and peri-infarct regions of MI rats. KANK2 lowered myocardial NF-κB p65 expression in MI rats. ConclusionKANK2 may play its therapeutic role in MI through improving cardiac function, decreasing myocardial collagen deposition, reducing cardiomyocyte apoptosis, and increasing angiogenesis, which might be associated with the reduction of NF-κB p65 expression.