Pro-inflammatory Cytokine Secretion Levels Research Articles

In Silico Studies: Stigmastan-3,6-dione in the Ethyl Acetate Fraction of Momordica charantia L Fruit has immunostimulant and anti-inflammatory activity

Momordica charantia L fruit provides an immunomodulatory effect by stimulating certain components of the body's immune system. Bioactive phytochemicals from M. charantia L function as anti-inflammatory agents by reducing levels of pro-inflammatory cytokine secretion including IL-1, IL-6, IL-8, TNF-α, NF-kB. This research looks at the insilico mechanism of action of the active ingredient of the EtOAc fraction of M. charantia L fruit as an immunostimulant and anti-inflammatory. The materials and methods used were an Intel Core i7 10 Th Gen laptop, Chem Office Professional 17.1, Chem 2D, Chem 3D software, PDB code (6W9K) and ligand (TUA), Stigmastan-3,6 dione (C29H48O2) from the EtOAc fraction of fruit M. charantia L was docked using MVD software, RMSD and RMSF values ​​were viewed using Yasara Software, pkCSM online tool to predict compound toxicity, toxicity prediction (LD50) was used by Protox online tool. The results of molecular docking of standard compounds, namely Methylprednisolone and Prednisolone and Stigmastan-3,6-dione, have Rerank Score values of -120.62, -121.47, -79.50, respectively. The lower the Rerank score value, the lower the binding energy between the protein and the ligand, causing the protein and ligand bonds to be more stable and it is predicted that the activity of the compound will be greater. The movement of RMSD values ​​between 0.6-1.9 Å for the 3 (three) compounds, is still within stable limits and does not undergo conformation. The RMSF value of the 3 (three) compounds has the same amino acid residue pattern. The insilico toxicity prediction for the 3 (three) compounds is still within safe limits. The EtOAc fraction of M. charantia L fruit with the active compound Stigmastan-3,6-Dione in its mechanism of action in silico shows activity as an anti-inflammatory and immunostimulant that works on the NF-kB pathway.

CpG Oligodeoxynucleotide-Coated Chitosan Nanoparticles Enhance Macrophage Proinflammatory Phenotype In Vitro.

This study aimed to investigate the effects of CpG Oligodeoxynucleotide (CpG ODNs)-Coated Chitosan Nanoparticles (CNP) on the phenotype of murine macrophages and their pro-inflammatory cytokine profile in vitro. CNP-CpG ODNs loaded with FITC-scrambled siRNA were prepared using the ionotropic gelation method. Peritoneal macrophages were isolated and exposed to CNP-CpG ODNs. Treated macrophages were assessed for uptake capacity. Flow cytometry was used to evaluate the expression levels of MHC-II, CD40, and CD86 costimulatory molecules in treated macrophages. Furthermore, the secretion levels of proinflammatory cytokines (TNF-α and IL-6) and the release of nitric oxide (NO) were measured in the culture supernatant of treated macrophages using sandwich ELISA and the Griess reaction, respectively. These in vitro studies showed that CNP-CpG ODNs had no cytotoxic effect on macrophages and were efficiently taken up by them. Additionally, CNP-CpG ODNs significantly increased the production of TNF-α, IL-6, and NO in the culture supernatant compared to CNP alone. Moreover, CNP-CpG ODNs enhanced the expression of MHC-II, CD40, and CD86 costimulatory molecules on macrophages. These findings indicate that incorporating CpG ODNs into CNPs promotes macrophage maturation and a proinflammatory phenotype. Therefore, CNP-CpG ODNs may serve as an effective system for targeted gene delivery to macrophages, enhancing immune responses.

Bushen Huoxue decotion-containing serum prevents chondrocyte pyroptosis in a m6A-dependent manner in facet joint osteoarthritis

BackgroundFacet joint osteoarthritis (FJOA) is a common lumbar osteoarthritis characterized by degeneration of small joint cartilage. Bushen Huoxue decotion (BSHXD) has good therapeutic effects on OA. Our work aimed to further probe the pharmacological effects of BSHXD-containing serum (BSHXD-CS) on FJOA and define underlying the mechanisms invovled. MethodsTo establish a FJOA cell model, primary rat chondrocytes were treated with LPS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. The secretion levels of pro-inflammatory cytokines were measured by ELISA. Cell viability was determined by CCK8 assay. The global m6A level was detected by the kit, and NLRP3 mRNA m6A level was determined by Me-RIP assay. The molecular interactions were analyzed by RIP and RNA pull-down assays. ResultsBSHXD-CS treatment relieved LPS-induced cell injury, inflammation, NLRP3 inflammasome and pyroptosis in chondrocytes (all p < 0.05). LPS-induced NLRP3 upregulation in chondrocytes was related to its high m6A modification level (p < 0.05). It was also observed that BSHXD-CS reduced LPS-induced m6A modification in chondrocytes via repressing STAT3 (all p < 0.05), suggesting BSHXD-CS could repress NLRP3 expression via m6A-dependent manner. Moreover, DAA, a m6A specific inhibitor, was proved to strengthen the protectively roles of BSHXD-CS on LPS-challenged pytoptosis (all p < 0.05). ConclusionBSHXD-CS inhibited NLRP3 inflammasome activation and pyroptosis in chondrocytes to repress OA progression by reducing RNA m6A modification.

Evaluation of antioxidant activity and fermentation properties of potential probiotic strain Lactiplantibacillus plantarum HY7720 in plant-based materials

People on vegan diets are at risk of being deficient in varied nutrients such as vitamin B12 and certain amino acids. In this study, we investigated vitamin B12-producing lactic acid bacteria (LAB) as well as the probiotic and antioxidant properties. Lactiplantibacillus plantarum HY7720 was screened from 22 strains of LAB that were isolated from different plant foods, and its growth ability and extracellular vitamin B12-producing capacity in vitamin B12-deficient medium were investigated. To determine whether HY7720 functions as a probiotic, survival rate in the simulated gastrointestinal tract and adhesion property to human intestinal epithelial cells of HY7720 were compared with positive control, Lacticaseibacillus rhamnosus GG (LGG). Moreover, the results showed that HY7720 recovered the gene expression levels of tight junction-associated proteins (TJPs), including TJP1, TJP2, occludin (OCLN), and claudin-1 (CLDN1) and inhibited the secretion levels of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-8, in tumor necrosis factor (TNF)-α-stimulated Caco-2 cells. Furthermore, we verified that HY7720 exhibit the antioxidant potential, by showing its intracellular reactive oxygen species (ROS) scavenging ability in hydrogen peroxide (H2O2)-stimulated Caco-2 cells. The ability of HY7720 to ameliorate H2O2-induced cytotoxicity in Caco-2 cells was inhibited by mitogen-activated protein kinase (MAPK) inhibitors, indicating that its antioxidant responses are related to extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK). This study also investigated the nutritional qualities of three plant-based materials (brown rice, white rice, and soy milk) fermented using HY7720. Collectively, HY7720 could be used as a promising probiotic strain for the prevention of nutritional deficiencies among people on vegetarian diets.

The expression of Nrf2 and TLRs in ear effusion in children with different types of otitis media and their relationship with inflammatory factors

ObjectiveThis study aimed to analyze the differences in the expression of Toll-like receptors (TLRs) and nuclear factor erythroid 2-related factor 2 (Nrf2) in ear effusion in children with different types of otitis media (OM), to elaborate the relationship between the expression of TLRs and Nrf2 in ear effusion and the pathogenesis of OM, and to explore the relationship between the two indicators and pro-inflammatory cytokines in children with OM, thereby laying a scientific foundation for revealing the underlying molecular mechanisms of the progression of different types of OM. MethodsA total of 73 children with OM who were treated in our hospital from March 2019 to July 2021 were selected as the study subjects. By using the cross-sectional investigation method, participants were divided into three groups according to the different pathological types, including the secretory OM group (30 cases), the chronic suppurative OM group (27 cases), and the cystic lesional OM group (16 cases). The levels of Nrf2, TLR2, TLR4 and proinflammatory cytokines [interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), transforming growth factor-β 1(TGF-β1), procalcitonin (PCT) and interleukin-1β (IL-1β)] were detected in ear effusion of children with different types of OM. Linear regression was used to analyze the correlation between the Nrf2, TLR2 and TLR4 expression levels and pro-inflammatory cytokines in ear effusion. ResultsThe expression levels of TNF-α and PCT in the ear effusion of the children under 3 years old were significantly higher than that of the children between 3 and 5 years old and that of the children between 6 and 8 years old (all P < 0.001). The mRNA levels of Nrf2, TLR2 and TLR4 in the ear effusion of the children from the chronic suppurative OM group were higher than these from the secretory OM group (P < 0.001, P = 0.008 and P = 0.021). The mRNA levels of Nrf2, TLR2, and TLR4 in the ear effusion of the children from the cystic lesional OM group were higher than those from the chronic suppurative OM group (P < 0.001, P = 0.029 and P = 0.018). A prominent increase in the concentrations of IFN-γ, TNF-α, TGF-β1, PCT and IL-1β was found in the ear effusion of children from the chronic suppurative OM group compared to these from the secretory OM group (P = 0.021, P = 0.044, P = 0.048, P = 0.004 and P = 0.001). The concentrations of IFN-γ, TNF-α, TGF-β1, PCT and IL-1β in the ear effusion of the children from the cystic lesional OM group were markedly increased as compared with these from the chronic suppurative OM group (P < 0.001, P = 0.004, P = 0.003, P < 0.001 and P < 0.001). Nrf2, TLR2 and TLR4 were taken as independent variables, and inflammatory indexes, including IFN-γ, TNF-α, TGF-β1, PCT and IL-1β were used as dependent variables for the linear regression analysis. The results showed that Nrf2, TLR2 and TLR4 were positively correlated with the secretion levels of pro-inflammatory cytokines after adjusting for age, sex, course and the OM classification (all P < 0.05). ConclusionThe expressions of Nrf2, TLR2 and TLR4 in the ear effusion of children with different types of OM gradually increased with the severity of the disease, these were significantly positively correlated with the pro-inflammatory cytokines of the children. Nrf2/TLR signaling pathway maintained chronic inflammation in OM, induced damage of middle ear tissue, and promoted the transition from acute OM to chronic OM.

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Effects of Moringa oleifera Seed Oil on Cultured Human Sebocytes In Vitro and Comparison with Other Oil Types.

The seeds of Moringa oleifera (horseradish tree) contain about 40% of one of the most stable vegetable oils (Moringa seed oil). Therefore, the effects of Moringa seed oil on human SZ95 sebocytes were investigated and were compared with other vegetable oils. Immortalized human SZ95 sebocytes were treated with Moringa seed oil, olive oil, sunflower oil, linoleic acid and oleic acid. Lipid droplets were visualized by Nile Red fluorescence, cytokine secretion via cytokine antibody array, cell viability with calcein-AM fluorescence, cell proliferation by real-time cell analysis, and fatty acids were determined by gas chromatography. Statistical analysis was performed by the Wilcoxon matched-pairs signed-rank test, the Kruskal-Wallis test and Dunn's multiple comparison test. The vegetable oils tested stimulated sebaceous lipogenesis in a concentration-dependent manner. The pattern of lipogenesis induced by Moringa seed oil and olive oil was comparable to lipogenesis stimulated by oleic acid with also similar fatty acid secretion and cell proliferation patterns. Sunflower oil induced the strongest lipogenesis among the tested oils and fatty acids. There were also differences in cytokine secretion, induced by treatment with different oils. Moringa seed oil and olive oil, but not sunflower oil, reduced the secretion of pro-inflammatory cytokines, in comparison to untreated cells, and exhibited a low n-6/n-3 index. The anti-inflammatory oleic acid detected in Moringa seed oil probably contributed to its low levels of pro-inflammatory cytokine secretion and induction of cell death. In conclusion, Moringa seed oil seems to concentrate several desired oil properties on sebocytes, such as high content level of the anti-inflammatory fatty acid oleic acid, induction of similar cell proliferation and lipogenesis patterns compared with oleic acid, lipogenesis with a low n-6/n-3 index and inhibition of secretion of pro-inflammatory cytokines. These properties characterize Moringa seed oil as an interesting nutrient and a promising ingredient in skin care products.

STING mediates SU5416/hypoxia-induced pulmonary arterial hypertension in rats by regulating macrophage NLRP3 inflammasome activation

BackgroundThe NLRP3 inflammasome in macrophages is known to promote infection-related vascular growth, and NLRP3 inflammasome activation interacts with PAH. STING is a crucial inflammatory reaction link that can increase the overexpression of NLRP3. However, the expression and effect of STING in PAH have not been elucidated. We examined the expression and articulation of STING in PAH and researched its hidden mechanism. MethodsA SU5416 plus hypoxia (Su/Hy)-induced rat model of PAH was constructed to examine STING activation. Su/Hy induced PAH rats were given a prophylactic injection of STING the inhibitor C-176. After modeling, hemodynamic changes, right ventricular hypertrophy index, lung morphological features, inflammasome activation, and proinflammatory cytokine secretion levels were assessed. In addition, the STING agonist DMXAA or inhibitor C-176 was used to interfere with LPS-induced BMDMs, NLRP3 inflammasome activation and cytokine secretion were examined, and the effect on PASMCs was evaluated in a coculture system. ResultsSTING expression increased significantly in the lung tissue of Su/Hy-treated PAH rats compared with normoxia-treated rats. Moreover, STING inhibitors can alleviate the Su/Hy-induced increase in pulmonary artery pressure and restrain the activation of the NLRP3 inflammasome and proinflammatory cytokines. In vitro experiments confirmed that STING affected the expression of the NLRP3 inflammasome and the secretion of inflammatory cytokines in BMDMs and promoted the proliferation of PASMCs in the coculture system. ConclusionOur study shows that STING is activated in Su/Hy-induced PAH and boosts the actuation of the macrophage NLRP3 inflammasome to advance the inflammatory response and vascular proliferation in rats with Su/Hy-induced pulmonary hypertension.

NLRP3 inflammasome activation and its inhibitory drugs in connection with COVID-19 infection

SARS-CoV-2 virus belongs to the beta coronavirus family that cause the inflammatory condition, acute pneumonia, and acute respiratory distress syndrome (ARDS). ARDS is the most important reason of mortality in patients, characterized as a highly increased levels of pro-inflammatory cytokine secretion. Inflammasome is a complex, which has an essential role in inflammatory situation, and NOD, LRR- and pyrin domain-containing protein 3 (NLRP3) is the most studied inflammasome that is considered to play vital roles in the virus infection and its pathogenesis. Our search language was limited to English and the search was performed in Web of Science, PubMed and Embase. Based on published articles, our current narrative review first explains the structure of the SARS-Cov2 virus and then describes the function of the NLRP3 inflammasome in relation to COVID-19 and drugs effective in controlling it. The NLRP3 inflammasome activation related to the initiation of inflammatory cascade including important cytokines production and releases such as IL-6, TNF-α and IL-1β. Thus, targeting the NLRP3 as a member of the innate immune system may be helpful for the reduction of ARDS clinical symptoms in COVID-19 patients.

Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-\u03baB pathways

BackgroundGiardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host’s innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored.MethodsIn this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs.ResultsThe results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects.ConclusionsThe results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions.Graphical abstract

Brilliant blue G attenuates neuro-inflammation via regulating MAPKs and NF-κB signaling pathways in lipopolysaccharide-induced BV2 microglia cells.

Previous studies have demonstrated that the P2X purinoceptor 7 (P2X7) receptor (P2X7R) serves a critical role in regulating the inflammatory response of various diseases in the central nervous system. The anti-inflammatory effect of brilliant blue G (BBG), a specific antagonist of the P2X7R, remains unclear in lipopolysaccharide (LPS)-induced BV-2 cells. The present study suggested that BBG attenuated the neuroinflammatory response; the protein levels of inducible oxide synthase and cyclooxygenase-2, and the mRNA and secretion levels of pro-inflammatory cytokines including interleukin (IL)-16, IL-1β and tumor necrosis factor-α (TNF-α), were all decreased in LPS-induced BV2 cells. BBG inhibited the activation of MAPKs by inhibiting the phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase and extracellular signal-regulated kinase. Notably, transcription factor p65 nuclear translocation was also inhibited, thereby leading to the inactivation of NF-κB. The inhibitory effects of BBG on MAPKs and NF-κB were additionally enhanced through the application of MAPK and NF-κB inhibitors. Taken together, the results demonstrated that BBG contributed to the suppression of the inflammatory effects in LPS-induced BV2 cells via the inhibition of NF-κB and MAPKs signaling pathways.

358 Toll-like receptor 2 partially dependent phagocytosis of cutibacterium acnes: Acne-associated strains phagocytosed more than healthy-associated strains

Pathogen phagocytosis is a fundamental process in innate immunity and important for host response to injury and infection. Some pathogenic bacteria possess structural or biochemical characteristics that allow them to resist or avoid host phagocytic and immune responses. Acne vulgaris is a multi-factorial inflammatory skin disorder with phagocytosis of Cutibacterium acnes (C. acnes) as an important part in disease pathogenesis. Previous studies have indicated Toll-like receptor 2 (TLR2) as a mediator of C. acnes-induced cytokine secretion, and other studies have observed immune cell phagocytic ability associated with TLR2 expression. In this study, we aimed to observe the effects of TLR2 functionality on C. acnes phagocytosis. Stimulation of TLR2-wild-type (WT) and TLR2-knock-out (KO) THP1 monocytes with C. acnes showed a decreased secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-12p70, and TNF-α in TLR2-KO cells. Flow cytometry analysis of TLR2-WT and TLR2-KO cells stimulated with fluorescently labeled C. acnes revealed decreased phagocytosis in TLR2-KO cells. However, phagocytosis was not completely prevented in TLR2-KO cells, suggesting involvement of other co-receptors in C. acnes phagocytosis. Single-cell RNA sequencing of acne lesions showed increased expression of ITGAM, ITGB2, and C5AR1 genes of the complement system when compared to non-lesional skin, suggesting that immune cell phagocytosis of C. acnes does not completely depend on TLR2 and may involve activation of the complement system. Further analysis revealed that in both TLR2-WT and TLR2-KO cells, monocytes phagocytosed more acne-skin associated C. acnes strains (CA) than healthy-skin associated strains (CH). While both CA and CH induce similar pro-inflammatory cytokine secretion levels, CA are phagocytosed more than CH, suggesting the possibility of a structural or biochemical difference between CA and CH that may influence immune cell pathogen recognition, phagocytosis, and anti-microbial activity.

PD47-07 A GENETICALLY MODIFIED BCG THAT OVEREXPRESSES A STING AGONIST SHOWS ENHANCED ANTITUMOR EFFICACY MEDIATED BY AN M1 MACROPHAGE POLARIZATION WITH POTENTIATION OF TRAINED IMMUNITY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV (PD47)1 Apr 2020PD47-07 A GENETICALLY MODIFIED BCG THAT OVEREXPRESSES A STING AGONIST SHOWS ENHANCED ANTITUMOR EFFICACY MEDIATED BY AN M1 MACROPHAGE POLARIZATION WITH POTENTIATION OF TRAINED IMMUNITY Alok Singh*, Monali Praharaj, Kara Lombardo, Andres Matoso, Gregory Joice, Takahiro Yoshida, Max Kates, David McConkey, William Bishai, and Trinity J. Bivalacqua Alok Singh*Alok Singh* More articles by this author , Monali PraharajMonali Praharaj More articles by this author , Kara LombardoKara Lombardo More articles by this author , Andres MatosoAndres Matoso More articles by this author , Gregory JoiceGregory Joice More articles by this author , Takahiro YoshidaTakahiro Yoshida More articles by this author , Max KatesMax Kates More articles by this author , David McConkeyDavid McConkey More articles by this author , William BishaiWilliam Bishai More articles by this author , and Trinity J. BivalacquaTrinity J. Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000934.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Trained immunity (TI) involving metabolic and epigenetic reprogramming of monocytes/macrophages confers a heightened immune status, and BCG has been shown to be a potent inducer of TI. We hypothesized that a genetically engineered BCG overexpressing a STING agonist may be a stronger inducer of antitumor TI. Using a recombinant BCG strain (BCG-disA-OE) which releases high levels of the STING agonist c-di-AMP, we tested induction of TI phenotypes in NMIBC models in vitro and in vivo. METHODS: WT-BCG (Tice strain) and BCG-disA-OE were compared for their induction of cytokines and phagocytic capacity in primary human and murine macrophages using ELISA, flow-cytometry, and immunofluorescence. We also measured epigenetic changes in the promoter regions of key cytokines using ChIP-PCR in human PBMC-derived monocytes. Antitumor efficacy was tested in the rat MNU-carcinogen model of NMIBC following intravesical BCG instillation weekly x 6 using histochemical tumor grading and qRT-PCR of bladder tissue. RESULTS: Compared with BCG-WT, BCG-disA-OE strains showed increased IFN I, IL-6, TNF-α, MCP-1 and IL-12 levels in primary macrophages and bladder cancer cell lines We observed increased M1 polarization shift and higher phagocytic capacity in primary macrophages after infection with rBCG-disA-OE. The modified BCG also elicited more pronounced epigenetic changes (H3K4Me3) on the TNF-α and IL-6 gene promoters following monocyte training. Intravesical instillations of BCG-disA-OE in MNU-rats resulted into significantly lower tumor involvement index and a more a potent induction of Th1 cytokines compared to BCG-WT. CONCLUSIONS: BCG-disA-OE demonstrated superior antitumor efficacy in the rat NMIBC model. It also elicited greater levels of pro-inflammatory cytokine secretion, M1 macrophage polarization, and epigenetic reprogramming. These observations suggest that BCG-disA-OE elicits more potent trained immunity changes monocytic cell populations in the bladder, and that elevated trained immunity may correlate with treatment outcome. BCG-disA-OE shows promise as a novel intravesical immunotherapy for NMIBC. Source of Funding: Maryland Tedco, Willowcroft Foundation, and Cigarette Restitution Fund © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e928-e928 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alok Singh* More articles by this author Monali Praharaj More articles by this author Kara Lombardo More articles by this author Andres Matoso More articles by this author Gregory Joice More articles by this author Takahiro Yoshida More articles by this author Max Kates More articles by this author David McConkey More articles by this author William Bishai More articles by this author Trinity J. Bivalacqua More articles by this author Expand All Advertisement PDF downloadLoading ...

Terminally Differentiated Effector Memory CD8+ T Cells Identify Kidney Transplant Recipients at High Risk of Graft Failure.

Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. We investigated the frequency and function of CD8+ T cell subsets-including effector memory (EM) and terminally differentiated EM (TEMRA) CD8+ T cells-in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8+ T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. Increased frequency of circulating TEMRA CD8+ T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8+ T cells associated with reduced risk of graft failure. A distinct TEMRA CD8+ T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8+ T cells, CD16 engagement resulted in selective activation of TEMRA CD8+ T cells, which mediated antibody-dependent cytotoxicity. At 1 year post-transplant, the composition of memory CD8+ T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8+ T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8+ T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8+ T cell monitoring for predicting risk of kidney transplant failure.

Clinically confirmed DEL-1 as a myokine attenuates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes via AMPK/HO-1- pathway

ABSTRACT Regular exercise is the first line of therapy for treating obesity-mediated metabolic disorders, including insulin resistance. It has been reported that developmental endothelial locus-1 (DEL-1) enhances macrophage efferocytosis, resulting in inflammation clearance as well as improves insulin resistance in skeletal muscle. However, the relationship between exercise and DEL-1, and the effects of DEL-1 on insulin signalling in adipocytes have not been fully elucidated to date. Protein expression levels were determined by Western blot analysis. Cells were transfected with small interfering (si) RNA to suppress gene expression. Lipid accumulation levels were detected using Oil red-O staining. Proinflammatory cytokine secretion levels were measured using ELISA. DEL-1 expression levels were induced in the skeletal muscle of people who exercised using microarray analysis. Recombinant DEL-1 augmented AMP-activated protein kinase (AMPK) phosphorylation and haem oxygenase (HO)-1 expression to alleviating inflammation and impairment of insulin signalling in 3T3-L1 adipocytes treated with palmitate. siRNA of AMPK or HO-1 also mitigated the effects of DEL-1 on inflammation and insulin resistance. DEL-1 ameliorates inflammation and insulin resistance in differentiated 3T3-L1 cells via AMPK/HO-1 signalling, suggesting that DEL-1 may be the exercise-mediated therapeutic target for treating insulin resistance and type 2 diabetes.

Immunomodulatory activities of zinc(II)phthalocyanine on the mammalian macrophages through p38 pathway: Potential ex vivo immunomodulatory PDT reagents

A series of zinc phthalocyanine having imidazolyl moieties was synthesized. These compounds’ immunostimulatory and immunomodulatory activities were tested on the mammalian macrophages in vitro. In the absence of photo induction neither dmso soluble nor the water soluble imidazole Pc had any immunostimulatory or immunomodulatory effect on the macrophage activity based on the differences in the pro-inflammatory cytokine secretion levels compared to the control groups. Upon photo induction, especially, at 5 min exposure time both derivatives lead to an increased pro-inflammatory cytokine secretion level by LPS activated macrophages. Whereas, this effect was completely reversed after 10 min of light treatment and both derivatives gained stark anti-inflammatory potential. Our molecules were cell penetrating and exerted their effects by regulating the phosphorylation levels of p38. This study is one of its first examples suggesting differential immunomodulatory photo dynamic therapy applications of phthalocyanine derivatives depending on light exposure time as well as their possible route of modulating the intracellular signaling pathways.

Characterization of the Cellular Responses of Dental Mesenchymal Stem Cells to the Immune System

IntroductionDental stem cells have gained importance recently and are being used for various purposes in regenerative medicine and dentistry. Although much research has been done to show the various properties of these dental stem cells, the immunomodulatory properties of some of these stem cells are still unknown. This is important considering these cells are being used routinely. Therefore, the aim of this study was to investigate the interactions between the activated immune cells and 3 types of dental-derived mesenchymal stem cells: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells of the apical papilla (SCAP). MethodsSCAP, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and periodontal ligament fibroblasts were cultured, and various assays were performed including a proliferation assay, flow cytometric analysis, lactate dehydrogenase and chromium-51 cytotoxicity assays, and an enzyme-linked immunosorbent assay to evaluate the interactions of these dental stem cells when cocultured with either peripheral blood mononuclear cells or natural killer cells. ResultsSCAP were less resistant to immune cell–mediated cytotoxicity as seen from the results obtained from the LDH and chromium-51 cytotoxicity assays. The flow cytometric analysis showed a lower resilience of SCAP to cytotoxic compounds. The enzyme-linked immunosorbent assay results demonstrated that the SCAP induced high levels of proinflammatory cytokine secretion compared with the other dental stem cells. ConclusionsSCAP did not perform as well as the other dental stem cells. This could in turn affect their survival and differentiation abilities as well as their functionality. This may be an important aspect to consider when selecting dental stem cells for various regenerative procedures.

Modulation of Macrophage Phenotype by Biodegradable Polyurethane Nanoparticles: Possible Relation between Macrophage Polarization and Immune Response of Nanoparticles.

Nanomaterials with surface functionalized by different chemical groups can either provoke or attenuate the immune responses of the nanomaterials, which is critical to their biomedical efficacies. In this study, we demonstrate that synthetic waterborne polyurethane nanoparticles (PU NPs) can inhibit the macrophage polarization toward the M1 phenotype but not M2 phenotype. The surface-functionalized PU NPs decrease the secretion levels of proinflammatory cytokines (TNF-α and IL-1β) for M1 macrophages. Specifically, PU NPs with carboxyl groups on the surface exhibit a greater extent of inhibition on M1 polarization than those with amine groups. These water-suspended PU NPs reduce the nuclear factor-κB (NF-κB) activation and suppress the subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome signals. Furthermore, the dried PU films assembled from PU NPs have a similar effect on macrophage polarization and present a smaller shifting foreign body reaction (FBR) in vivo than the conventional poly(l-lactic acid). Taken together, the biodegradable waterborne PU NPs demonstrate surface-dependent immunosuppressive properties and macrophage polarization effects. The findings suggest potential therapeutic applications of PU NPs in anti-inflammation and macrophage-related disorders and propose a mechanism for the low FBR observed for biodegradable PU materials.

Apigenin Retards Atherogenesis by Promoting ABCA1-Mediated Cholesterol Efflux and Suppressing Inflammation

Background/Aims: The development of atherosclerosis is accompanied by escalating inflammation and lipid accumulation within blood vessel walls. ABCA1 plays a crucial role in mediating cholesterol efflux from macrophages, which protects against atherogenesis. This research was designed to explore the effects and underlying mechanisms of apigenin (4’, 5, 7-trihydroxyflavone) on ABCA1-mediated cellular cholesterol efflux and LPS-stimulated inflammation in RAW264.7 macrophages and apoE^-/- mice. Methods: Expression of genes or proteins was examined by RT-PCR or western blot analysis. Liquid scintillation counting was used to detect percent cholesterol efflux. Cellular cholesterol content was measured using HPLC assay. The secretion levels of pro-inflammatory cytokines were quantified by ELISA assay. Atherosclerotic lesion sizes were determined with Oil Red O staining. The contents of macrophages and smooth muscle cells in atherosclerotic lesion were evaluated using immunohistochemistry. Plasma TC, TG, HDL-C and LDL-C levels in apoE^-/- mice were evaluated using commercial test kits. Results: Apigenin potently increased ABCA1 expression through miR-33 repression in a dose- and time-dependent manner. Treatment with apigenin significantly increased ABCA1-mediated cholesterol efflux, and reduced TC, FC and CE levels in macrophage-derived foam cells. In LPS-treated macrophages, the expression levels of TLR-4, MyD88 and p-IκB-α as well as nuclear NF-κB p65 were decreased by the addition of apigenin. Moreover, apigenin markedly decreased secretion levels of several pro-inflammatory cytokines. Lastly, in LPS-challenged apoE^-/- mice, apigenin administration augmented ABCA1 expression, decreased the contents of macrophages and smooth muscle cells in atherosclerotic lesion, reduced miR-33, TLR-4, and NF-κB p65 levels, improved plasma lipid profile and relieved inflammation, which results in less atherosclerotic lesion size. Conclusions: Taken together, these results suggest that apigenin may attenuate atherogenesis through up-regulating ABCA1-mediated cholesterol efflux and inhibiting inflammation.

Correlation of CDC42 Activity with Cell Proliferation and Palmitate-Mediated Cell Death in Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stromal Cells.

RHO GTPases regulate cell migration, cell-cycle progression, and cell survival in response to extracellular stimuli. However, the regulatory effects of RHO GTPases in mesenchymal stromal cells (MSCs) are unclear. Herein, we show that CDC42 acts as an essential factor in regulating cell proliferation and also takes part in lipotoxic effects of palmitate in human umbilical cord Wharton's jelly derived MSCs (hWJ-MSCs). Cultured human bone marrow, adipose tissue, and hWJ-MSC derived cells had varying pro-inflammatory cytokine secretion levels and cell death rates when treated by palmitate. Strikingly, the proliferation rate of these types of MSCs correlated with their sensitivity to palmitate. A glutathione-S-transferase pull-down assay demonstrated that hWJ-MSCs had the highest activation of CDC42, which was increased by palmitate treatment in a time-dependent manner. We demonstrated that palmitate-induced synthesis of pro-inflammatory cytokines and cell death was attenuated by shRNA against CDC42. In CDC42 depleted hWJ-MSCs, population-doubling levels were notably decreased, and phosphorylation of ERK1/2 and p38 MAPK was reduced. Our data therefore suggest a mechanistic role for CDC42 activity in hWJ-MSC proliferation and identified CDC42 activity as a promising pharmacological target for ameliorating lipotoxic cell dysfunction and death.