LINC01686 affects LPS-induced cytokine expression via the miR-18a-5p/A20/STAT1 axis in THP-1 cells.

BACKGROUND AND OBJECTIVE: Long noncoding RNAs (lncRNAs) are crucial in regulating various physiological and pathological processes, including immune responses. LINC01686 is a lncRNA with previously uncharacterized functions in immune regulation. This study aims to investigate the function of LINC01686 in lipopolysaccharide (LPS)-induced inflammatory responses in the human monocytic leukemia cell line THP-1 and its potential regulatory mechanisms involving miR-18a-5p and the anti-inflammatory protein A20. METHOD: THP-1 cells were stimulated with LPS to induce inflammatory responses, followed by analysis of LINC01686 expression levels. The role of LINC01686 in regulating the expression of interleukin (IL)-6, IL-8, A20, and signal transducer and activator of transcription 1 (STAT1) was examined using small interfering RNA-mediated knockdown. Additionally, the involvement of miR-18a-5p in LINC01686-mediated regulatory pathways was assessed by transfection with decoy RNAs mimicking the miR-18a-5p binding sites of LINC01686 or A20 messenger RNA. RESULTS: LINC01686 expression was upregulated in THP-1 cells following LPS stimulation. Suppression of LINC01686 enhanced LPS-induced expression of IL-6 and IL-8, mediated through increased production of reactive oxygen species. Moreover, LINC01686 knockdown upregulated the expression and activation of IκB-ζ, STAT1, and downregulated A20 expression. Transfection with decoy RNAs reversed the effects of LINC01686 suppression on A20, STAT1, IL-6, and IL-8 expression, highlighting the role of LINC01686 in sponging miR-18a-5p and regulating A20 expression. CONCLUSION: This study provides the first evidence that LINC01686 plays a critical role in modulating LPS-induced inflammatory responses in THP-1 cells by sponging miR-18a-5p, thereby regulating the expression and activation of A20 and STAT1. These findings shed light on the complex regulatory mechanisms involving lncRNAs in immune responses and offer potential therapeutic targets for inflammatory diseases.

Establishment of an in vitro model of monocyte-like THP-1 cells for trained immunity induced by bacillus Calmette-Guérin.

BACKGROUND: Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. RESULTS: In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1ß) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed. CONCLUSIONS: We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.

Macrophage-derived exosomal miRNA-141 triggers endothelial cell pyroptosis by targeting NLRP3 to accelerate sepsis progression.

Sepsis, critical condition marked by severe organ dysfunction from uncontrolled infection, involves the endothelium significantly. Macrophages, through paracrine actions, play a vital role in sepsis, but their mechanisms in sepsis pathogenesis remain elusive. Objective: We aimed to explore how macrophage-derived exosomes with low miR-141 expression promote pyroptosis in endothelial cells (ECs). Exosomes from THP-1 cell supernatant were isolated and characterized. The effects of miR-141 mimic/inhibitor on apoptosis, proliferation, and invasion of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed using flow cytometry, CCK-8, and transwell assays. Key pyroptosis-related proteins, including caspase-1, IL-18, IL-1ß, NLR Family Pyrin Domain Containing 3 (NLRP3), ASC, and cleaved-GSDMD, were analyzed via Western blot. The interaction between miR-141 and NLRP3 was studied using RNAhybrid v2.2 and dual-Luciferase reporter assays. The mRNA and protein level of NLRP3 after exosomal miR-141 inhibitor treatment was detected by qPCR and Western blot, respectively. Exosomes were successfully isolated. miR-141 mimic reduced cell death and pyroptosis-related protein expression in HUVECs, while the inhibitor had opposite effects, increasing cell death, and enhancing pyroptosis protein expression. Additionally, macrophage-derived exosomal miR-141 inhibitor increased cell death and pyroptosis-related proteins in HUVECs. miR-141 inhibits NLRP3 transcription. Macrophages facilitate sepsis progression by secreting miR-141 decreased exosomes to activate NLRP3-mediated pyroptosis in ECs, which could be a potentially valuable target of sepsis therapy.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

The S1 spike protein of SARS-CoV-2 upregulates the ERK/MAPK signaling pathway in DC-SIGN-expressing THP-1 cells.

Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylated bovine serum albumin, the serum amyloid beta protein, and intracellular adhesion molecule 3 suppressed ERK phosphorylation, suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.

Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

Objective: Bi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD. Methods: Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity. Results: Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures. Conclusion: MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.

Saharan dust induces the lung disease-related cytokines granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

Desert dust exposure is associated with adverse respiratory health effects. Desert dust is a complex pollutant mixtures that includes respirable crystalline and amorphous particles, metals, and microbial constituents. Given the health effects of desert dust and its heterogeneity, as yet unidentified harmful biological pathways may be triggered. Therefore, we exposed human in vitro air-liquid interface co-cultures of alveolar epithelial A549 cells and THP-1 macrophages to Saharan dust (SD). For comparison, we used the known pulmonary toxicant DQ12 quartz dust. Via RNA sequencing, we identified that SD but not DQ12 increased the gene expression of granulocyte-macrophage colony-stimulating factor (GMCSF) and granulocyte colony-stimulating factor (GCSF). These findings were confirmed by quantitative reverse transcriptase PCR. SD dose-dependently upregulated GMCSF and GCSF expression with significant 7 and 9-fold changes, respectively, at the highest tested concentration of 31 µg/cm2. Furthermore, we observed that SD significantly enhanced the secretion of GM-CSF and G-CSF by 2-fold. Both cytokines have previously been associated with lung diseases such as asthma and fibrosis. Hence, we present two molecular messengers that may contribute to the adverse health effects of desert dust and might serve as drug targets for this globally relevant non-anthropogenic air pollutant.

Astaxanthin targets IL-6 and alleviates the LPS-induced adverse inflammatory response of macrophages.

Numerous natural compounds are recognized for their anti-inflammatory properties attributed to antioxidant effects and the modulation of key inflammatory factors. Among them, astaxanthin (AST), a potent carotenoid antioxidant, remains relatively underexplored regarding its anti-inflammatory mechanisms and specific molecular targets. In this study, human monocytic leukemia cell-derived macrophages (THP-1) were selected as experimental cells, and lipopolysaccharides (LPS) served as inflammatory stimuli. Upon LPS treatment, the oxidative stress was significantly increased, accompanied by remarkable cellular damage. Moreover, LPSs escalated the expression of inflammation-related molecules. Our results demonstrate that AST intervention could effectively alleviate LPS-induced oxidative stress, facilitate cellular repair, and significantly attenuate inflammation. Further exploration of the anti-inflammatory mechanism revealed AST could substantially inhibit NF-κB translocation and activation, and mitigate inflammatory factor production by hindering NF-κB through the antioxidant mechanism. We further confirmed that AST exhibited protective effects against cell damage and reduced the injury from inflammatory cytokines by activating p53 and inhibiting STAT3. In addition, utilizing network pharmacology and in silico calculations based on molecular docking, molecular dynamics simulation, we identified interleukin-6 (IL-6) as a prominent core target of AST anti-inflammation, which was further validated by the RNA interference experiment. This IL-6 binding capacity actually enabled AST to curb the positive feedback loop of inflammatory factors, averting the onset of possible inflammatory storms. Therefore, this study offers a new possibility for the application and development of astaxanthin as a popular dietary supplement of anti-inflammatory or immunomodulatory function.

4-(Benzyloxy)phenol-induced p53 exhibits antimycobacterial response triggering phagosome-lysosome fusion through ROS-dependent intracellular Ca2+ pathway in THP-1 cells.

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 µM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.

HDAC11 Regulates Palmitate-induced NLRP3 Inflammasome Activation by Inducing YAP Expression in THP-1 Cells and PBMCs.

Histone deacetylase 11 (HDAC11) has been implicated in the pathogenesis of metabolic diseases characterized by chronic low-grade inflammation, such as obesity. However, the influence of HDAC11 on inflammation and the specific effect of HDAC11 on the palmitic acid (PA)-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation are poorly understood. The effect of PA treatment on HDAC11 activity and the NLRP3 inflammasome was investigated in human peripheral blood mononuclear cells and THP-1 cells. The PA-induced responses of key markers of NLRP3 inflammasome activation, including NLRP3 gene expression, caspase-1 p10 activation, cleaved IL-1ß production, and extracellular IL-1ß release, were assessed as well. The role of HDAC11 was explored using a specific inhibitor of HDAC11 and by knockdown using small interfering (si)HDAC11 RNA. The relationship between HDAC11 and yes-associated protein (YAP) in the PA-induced NLRP3 inflammasome was investigated in THP-1 cells with HDAC11 or YAP knockdown. Following PA treatment, HDAC11 activity and protein levels increased significantly, concomitant with activation of the NLRP3 inflammasome. Notably, PA-induced the upregulation of NLRP3, caspase-1 p10 activation, the production of cleaved IL-1ß, and the release of IL-1ß into the extracellular space, all of which were attenuated by FT895 treatment and by HDAC11 knockdown. In THP-1 cells, PA induced the expression of YAP and its interaction with NLRP3, resulting in NLRP3 inflammasome activation, whereas both were inhibited by FT895 and siHDAC11 RNA. These findings demonstrate a pivotal role for HDAC11 in the PA-induced activation of the NLRP3 inflammasome. HDAC11 inhibition thus represents a promising therapeutic strategy for mitigating NLRP3 inflammasome-related inflammation in the context of obesity.

Difference in the cellular response following THP-1 derived phagocytic monocyte cells exposure to commercial aluminum-based adjuvants and aluminum-containing vaccines.

BACKGROUND: Aluminum-based adjuvants (ABAs) enhance the immune response following vaccine injection. Their mechanisms of action are not fully understood, and their bio-persistency have been described associated with long-term adverse effects. METHODS: We evaluated and compared the cellular effects of the two main ABAs and whole vaccines on ATP production, ROS generation and cytokines production (IL-6 and IL-10), using THP-1 cells. RESULTS: ABAs altered the cell energy metabolism by increasing ROS production after 24 h and reducing ATP production after 48 h. In addition, both ABAs and whole vaccines induced different kinetics of IL-6 production, whereas only ABAs induced IL-10 secretion. CONCLUSION: This study showed clearly, for a first time, a difference in cellular response to the ABAs and whole vaccines which should be taken into consideration in future studies focusing on the effect of ABA in vaccines. Future studies on ABAs should also pay attention to mitochondrial function alterations following exposure to ABA-containing vaccines.

Intracellular Protein Adsorption Behavior and Biological Effects of Polystyrene Nanoplastics in THP-1 Cells.

Micro(nano)plastics (MNPs) are emerging pollutants that can adsorb pollutants in the environment and biological molecules and ultimately affect human health. However, the aspects of adsorption of intracellular proteins onto MNPs and its biological effects in cells have not been investigated to date. The present study revealed that 100 nm polystyrene nanoplastics (NPs) could be internalized by THP-1 cells and specifically adsorbed intracellular proteins. In total, 773 proteins adsorbed onto NPs with high reliability were identified using the proteomics approach and analyzed via bioinformatics to predict the route and distribution of NPs following cellular internalization. The representative proteins identified via the Kyoto Encyclopedia of Genes and Genomes pathway analysis were further investigated to characterize protein adsorption onto NPs and its biological effects. The analysis revealed that NPs affect glycolysis through pyruvate kinase M (PKM) adsorption, trigger the unfolded protein response through the adsorption of ribophorin 1 (RPN1) and heat shock 70 protein 8 (HSPA8), and are chiefly internalized into cells through clathrin-mediated endocytosis with concomitant clathrin heavy chain (CLTC) adsorption. Therefore, this work provides new insights and research strategies for the study of the biological effects caused by NPs.

MiR-548t-5p regulates pancreatic ductal adenocarcinoma metastasis through an IL-33-dependent crosstalk between cancer cells and M2 macrophages.

IL-33 has been associated with pro- and anticancer functions in cancer. However, its role in pancreatic cancer metastasis remains unknown. This study aimed to explore the role of miR-548t-5p/IL-33 axis in the metastasis of pancreatic cancer. Luciferase activity assay, qRT-PCR, Western blot and ELISA were performed to prove whether IL-33 is the target of miR-548t-5p. In vivo metastasis assay and cellular transwell assay were performed to explore the role of miR-548t-5p/IL-33 axis in the invasion and metastasis of pancreatic cancer. Co-culture experiments and immunohistochemistry were performed to observe whether IL-33 affects cell invasion and metastasis dependent on the involvement of M2 macrophages. THP-1 cell induction experiment and flow cytometry were performed to explore the effect of IL-33 on macrophage polarization. CCK-8, colony formation, cell apoptosis, cell cycle, cell wound healing and transwell assay were performed to investigate the effect of IL-33 induced M2 macrophages on cell malignant biological behavior by coculturing pancreatic cancer cells with the conditioned medium (CM) from macrophages. We found that miR-548t-5p regulated the expression and secretion of IL-33 in pancreatic cancer cells by directly targeting IL-33 mRNA. IL-33 secreted by cancer cells promoted the recruitment and activation of macrophages to a M2-like phenotype. In turn, IL-33 induced M2 macrophages promoted the migration and invasion of cancer cells. Moreover, IL-33 affected pancreatic cancer cell invasion dependent on the involvement of M2 macrophages in the co-culture system. Thus, our study suggested that manipulation of this IL-33-dependent crosstalk has a therapeutic potential for the treatment of pancreatic cancer metastasis.

Differentiation, maturation, and collection of THP-1-derived dendritic cells based on a PEG hydrogel culture platform.

PURPOSE: Dendritic cell (DC) is a spearhead responsible for immune response and surrounded by extracellular matrix in three-dimensional (3D) tissue. Nevertheless, conventional DC culture has relied on suspension or two-dimensional (2D) tissue culture plate (TCP)-based culture system. This culture condition often fails to recapitulate the physiological behavior of DC in real tissue. In this work, the effect of culture condition on DC physiology was explored with varying 3D hydrogel property (i.e., degradability, adhesion, and stiffness). In particular, DC differentiation and maturation in 3D were evaluated comparing the conventional TCP-based culture condition. METHOD: THP-1 cells were encapsulated in poly(ethylene glycol) (PEG) hydrogel via thiol-ene photocrosslinking with non-degradable or proteolytically degradable peptide crosslinker. Hydrogel stiffness was manipulated by controlling the concentration of crosslinker. The metabolic activities and cytotoxicity of the encapsulated cells were measured by resazurin and Live/Dead assays, respectively. Cell harvesting was conducted via enzymatic degradation using α-chymotrypsin, and differentiation and maturation of the liberated DCs were evaluated by quantitative polymerase chain reaction and flow cytometry. RESULTS: THP-1 cells well proliferated in the soft degradable hydrogel with a higher metabolic activity. However, the stiff matrix inhibited cell growth in 3D. The gene expression assay indicated that the 3D hydrogel condition was superior to 2D culture in terms of differentiation and maturation of DC. Interestingly, the stiffness of matrix was important factor in DC function. In the stiff hydrogel, the expression levels of differentiation and maturation markers were higher compared to the low stiffness hydrogel. The mature DCs caged in the hydrogel matrix were harvested after short enzymatic digestion of hydrogel and the liberated cells had over 90% viability. The flow cytometric result revealed that the proportion of CD80 + /CD86 + cells from the stiff hydrogel was relatively higher than cells either from 2D or soft hydrogel in 3D. CONCLUSION: The collected evidence indicated that the proteolytically degradable PEG hydrogel matrix promoted DC differentiation and maturation. In addition, the matrix stiffness control could manipulate the marker expressions of differentiation and maturation. Particularly, the mature DC was successfully collected from the hydrogel matrix. These results highlighted the PEG hydrogel-based DC culture might be a useful tool for potential DC-based immunotherapies.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

BACKGROUND: Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. OBJECTIVE: In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. METHODS: Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1ß, Transforming Growth Factor (TGF ß1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene ß-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. RESULTS: The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. CONCLUSIONS: Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

The exoprotein Gbp of Fusobacterium nucleatum promotes THP-1 cell lipid deposition by binding to CypA and activating PI3K-AKT/MAPK/NF-κB pathways.

INTRODUCTION: Growing evidence has shown the correlation between periodontitis and atherosclerosis, while our knowledge on the pathogenesis of periodontitis-promoting atherosclerosis is far from sufficient. OBJECTIVES: Illuminate the pathogenic effects of Fusobacterium nucleatum (F. nucleatum) on intracellular lipid deposition in THP-1-derived macrophages and elucidate the underlying pathogenic mechanism of how F. nucleatum promoting atherosclerosis. METHODS AND RESULTS: F. nucleatum was frequently detected in different kinds of atherosclerotic plaques and its abundance was positively correlated with the proportion of macrophages. In vitro assays showed F. nucleatum could adhere to and invade THP-1 cells, and survive continuously in macrophages for 24 h. F. nucleatum stimulation alone could significantly promote cellular inflammation, lipid uptake and inhibit lipid outflow. The dynamic gene expression of THP-1 cells demonstrated that F. nucleatum could time-serially induce the over-expression of multiple inflammatory related genes and activate NF-κB, MAPK and PI3K-AKT signaling pathways. The exoprotein of F. nucleatum, D-galactose-binding protein (Gbp), acted as one of the main pathogenic proteins to interact with the Cyclophilin A (CypA) of THP-1 cells and induced the activation of the NF- κB, MAPK and PI3K-AKT signaling pathways. Furthermore, use of six candidate drugs targeting to the key proteins in NF- κB, MAPK and PI3K-AKT pathways could dramatically decrease F. nucleatum induced inflammation and lipid deposition in THP-1 cells. CONCLUSIONS: This study suggests that the periodontal pathogen F. nucleatum can activate macrophage PI3K-AKT/MAPK/NF-κB signal pathways, promotes inflammation, enhances cholesterol uptake, reduces lipid excretion, and promotes lipid deposition, which may be one of its main strategies promoting the development of atherosclerosis.

ALKBH5 promotes the development of lung adenocarcinoma by regulating the polarization of M2 macrophages through CDCA4.

OBJECTIVE: Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer, with high morbidity and mortality. N6-methyladenosine (m6A) is an important regulator of LUAD progression. Here, we investigated the potential biological functions of ALKBH5 (a m6A demethylated enzyme) and cell division cycle associated protein 4 (CDCA4) in the progression of LUAD. METHODS: The expressions of CDCA4, METTL3, ALKBH5, FTO, YTHDC2 and YTHDC1 mRNA and proteins in LUAD and adjacent tissues, as well as NCI-H1299 and NCI-H157 cells were detected by RT-qPCR and western blot. Meanwhile, the role of ALKBH5 and CDCA4 in macrophage polarization was explored through tumor formation in Lewis lung carcinoma (LLC) mice and the co-culture system of NCI-H1299 and NCI-H157/THP-1 cells. Cell characterization was further analyzed. The expression of Ki-67 in tumor tissue was tested by immunohistochemistry. The scale of M1 and M2 macrophages was determined by flow cytometry. RESULTS: CDCA4 was significantly overexpressed in NCI-H1299 and NCI-H157 cell lines compared with BEAS-2B cells. The fold enrichment of CDCA4 m6A level in the overexpression (oe)-METTL3 or short hairpin (sh)-ALKBH5 cells was enhanced. Overexpression of CDCA4 promoted the cell viability, proliferation and migration, and inhibited apoptosis, which was reversed by sh-ALKBH5 intervention. Overexpression of YTHDC2 (not YTHDC1) inhibited the effect of CDCA4 on sh-ALKBH5 cells. sh-CDCA4 inhibited tumor growth and weight of LLC cells in mice, and promoted M1/M2 ratio in LLC mice and NCI-H1299/THP-1 and NCI-H157/THP-1 co-culture systems. Oe-CDCA4 promoted the volume and weight of tumor and inhibited the M1/M2 ratio of tumor tissue in LLC mice, but was reversed by sh-ALKBH5 intervention. CONCLUSION: m6A demethylase ALKBH5 promotes the development of LUAD through CDCA4 regulation of malignant characterization and M1/M2 macrophage polarization.

Screening Allergenic Potencies of Skin-Contact Products Using the Human-Derived THP-1 Cell Activation Test.

With the prevalence of allergic contact dermatitis (ACD) from the usage of skin-contact products, like wearable, skin care, and hair care products, screening their skin sensitizing potential is necessary, for the sake of alleviating the consequent public health impact. In the present study, a total of 77 skin-contact products classified by four categories, watch bands (WBs), skin care products (SCPs), hair care products (HCPs), and rubber gloves (RGs), were investigated, using an optimized in vitro assay of human cell line activation test (h-CLAT). Extracting the products using neutral artificial sweat simulated well the practical usage scenarios, and testing the extracts showed that 26 of them were allergy test positive, including nine WBs, six SCPs, two HCPs, and nine RGs. The allergenic response was mainly characterized by the induction of CD54 expression, and diverse paradigms of CD54 and CD86 levels were observed by analyzing dose-response curves, which could also be influenced by the compromised viability of the THP-1 cells. The data implicated the intricate regulation by different contributors to suspicious ingredients in the test samples. Altogether, a promising methodology for testing skin allergy potential was well established for commonly used commodities by neutral artificial sweat extraction coupled with h-CLAT screening. The findings would be of great help in tracing the potential allergens in practical products and improving their qualities.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

[The Protective Action of Hsp70 and Hydrogen Sulfide Donors in THP-1 Macrophages in the Lipopolysaccharide-Induced Inflammatory Response by Modulating Endocytosis].

Hsp70 and hydrogen sulfide donors reduce inflammatory processes in human and animal cells. The biological action mediated by Hsp70 and H2S donors (GYY4137 and sodium thiosulfate) depends on their protection kinetics from cell activation by lipopolysaccharides. However, the molecular mechanisms of action of Hsp70 and H2S are not well understood. We studied the effect of human recombinant Hsp70 and H2S donors on the formation of reactive oxygen species and tumor necrosis factor-alpha induced in human cells (THP-1) by lipopolysaccharides. Transcriptomic changes occurring in these cells after LPS administration in combination with GYY4137 pretreatment were investigated. The results we obtained showed that Hsp70 and hydrogen sulfide donors reduce inflammatory processes in cells activated by the action of LPS. Hsp70 and H2S donors differed in the kinetics of the protective action, while hydrogen sulfide donors turned out to be more effective. The role of endocytosis in the mechanisms of protection of cells by H2S and Hsp70 donors from the action of LPS was studied. It has been found that GYY4137 pretreatment of LPS-exposed cells reduces the LPS-induced induction of various pro-inflammatory genes and affects the expression of genes of various intracellular signaling pathways.