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Human monocytes and dendritic cells express transient receptor potential vanilloid 1 (TRPV1) which may play a role in mediating the inflammatory, immune and cancer surveillance responses of these cells. The aim of the present study was to investigate TRPV1 expression and function in THP-1 monocytic cells. RT-PCR and Western blot were used to detect TRPV1. The metabolic activity and viability of THP-1 cells following exposure to vanilloids was assessed using resorufin production from rezazurin. Cytokine release was measured using ELISA. TRPV1 was expressed in cultured THP-1 monocytic cells and naïve monocytes. Lower concentrations (250 μM) of capsaicin, but not other putative TRPV1 agonists, were shown to stimulate cell metabolic activity, whereas at concentrations250 μM, all agonists decreased metabolic activity. Capsaicin-stimulated THP-1 metabolic activity was blocked by the TRPV1 antagonist, 5-iodo-resiniferatoxin (5'-IRTX), whereas the decline in resorufin production by THP-1 cells at higher capsaicin concentrations (due to cell death), was not affected by 5'-IRTX. Finally, capsaicin (≤125 μM) significantly increased lipopolysaccharide-stimulated IL-6 and TNF-α release from THP-1 cells, whereas phytohaemagglutinin-stimulated IL-1β, TNF-α, MCP-1 and IL-6 release were concentration-dependently inhibited by capsaicin. Modulation of IL-1β release was not TRPV1 mediated. Overall, these results show that functional TRPV1 channels are present in naïve monocytes and THP-1 cells, and when activated, increase cell metabolic activity. In addition, capsaicin modifies cytokine release from THP-1 cells and induces cell death, most likely by a mechanism that is independent of TRPV1 activation.

Macrophages are paramount to the initiation and procession of atherosclerosis, thus targeting macrophages in the progress of atherosclerosis is indispensable. Therefore, we perform in vitro experiments to investigate the effects of FGF-21 on macrophages in the progress of atherosclerosis. First, we use phorbol-12-myristate-13-acetate (PMA), a phorbol ester, to induce THP-1 cells into macrophages as macrophages model. After that we use Ox-LDL to induce macrophages into foam cells and simultaneously administrate with FGF-21 or not to determine whether FGF-21 has effects on foam cells formation and related inflammatory response. Wound healing results show that FGF-21 can inhibit macrophage migration. Oil Red-O stain, immunofluorescence and flow cytometer results show that FGF-21 can repress cholesterol accumulation in macrophages thereby inhibit foam cells formation and these effects can be abolished by FGFR inhibitor. Moreover, real-time PCR results showed that FGF-21 significantly reduces expression of inflammatory factors including IL-1α, IL-6 and TNF-α and this effect can be abolished by FGFR inhibitor. Furthermore, to determine the mechanism of FGF-21 regulates inflammatory response in Ox-LDL-induced THP-1 macrophages, western blotting results show that after treatment of Ox-LDL in macrophages, NF-κB signaling pathway is activated but FGF-21 can significantly inhibit this pathway. In addition, FGF-21 also regulates some regulators of lipid metabolism after treatment of Ox-LDL in macrophages. Above all, our findings demonstrate that FGF-21 can regulate foam cells formation, macrophage migration, inflammatory response and lipid metabolism in Ox-LDL-induced THP-1 macrophages.

Environmental exposure to arsenic is known to induce immunotoxicity. Macrophages are the professional phagocytes that are important in the immune system. In this study, we utilized the macrophages derived from the THP-1 human monocyte cell line as the experimental model to study the functional suppression induced by arsenite (As+3), one of the most prevalent forms of inorganic arsenic, at environmentally-relevant concentrations. Apoptosis was observed in the THP-1 derived macrophages treated with 500 nM As+3 for 18 h. Suppression of phagocytosis was induced by 18 h As+3 treatment starting from 100 nM. Suppressive effects on the production of two pro-inflammatory cytokines, IL-1β and TNF-α, were also found with the treatment of low to moderate doses of As+3 in lipopolysaccharides-stimulated THP-1 derived macrophages. The nitric oxide production was also inhibited by As+3 treatments, which was negatively correlated with the production of superoxide. Collectively, the results from the study demonstrated that environmentally-relevant concentrations of As+3 induced cytotoxicity and suppressed the major cellular functions in THP-1 derived macrophages. The macrophages were showed to be relatively sensitive to As+3, and could be the essential target of the toxicity induced by environmental arsenic exposures.

Graphical abstract, Hypertension has been recognized as one of the most frequent comorbidities and risk factors for the seriousness and adverse consequences in COVID-19 patients. 3,4-dihydropyrimidin-2(1H) ones have attracted researchers to be synthesized via Beginilli reaction and evaluate their antihypertensive activities as bioisosteres of nifedipine a well-known calcium channel blocker. In this study, we report synthesis of some bioisosteres of pyrimidines as novel CCBs with potential ACE2 inhibitory effect as antihypertensive agents with protective effect against COVID-19 infection by suppression of ACE2 binding to SARS-CoV-2 Spike RBD. All compounds were evaluated for their antihypertensive and calcium channel blocking activities using nifedipine as a reference standard. Furthermore, they were screened for their ACE2 inhibition potential in addition to their anti‐inflammatory effects on LPS-stimulated THP‐1 cells. Most of the tested compounds exhibited significant antihypertensive activity, where compounds 7a, 8a and 9a exhibited the highest activity compared to nifedipine. Moreover, compounds 4a,b, 5a,b, 7a,b, 8a,c and 9a showed promising ACE2:SARS-CoV-2 Spike RBD inhibitory effect. Finally, compounds 5a, 7b and 9a exerted a promising anti-inflammatory effect by inhibition of CRP and IL-6 production. Ultimately, compound 9a may be a promising antihypertensive candidate with anti-inflammatory and potential efficacy against COVID-19 via ACE2 receptor inhibition.

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.

Objective This study aimed to investigate the expression of long non-coding RNA (lncRNA) growth arrest-special transcript 5 (GAS5) in the serum of tuberculosis (TB) patients and discuss the mechanism of GAS5 in TB by establishing an in-vitro TB cell model. Methods Serum expressions of GAS5 and miR-18a-5p were determined by quantitative real-time PCR (qRT-PCR). The effects of GAS5 on macrophage cell viability and the inflammatory response after MTB infection were assessed by CCK-8 and ELISA. Luciferase reporter gene assay was applied to delve into the potential target gene of GAS5. Results The expression of GAS5 in TB patients was down-regulated, while miR-18a-5p was up-regulated, and the serum inflammatory factors were negatively correlated with the expression level of GAS5. MTB infection induced significant upregulation on the cell viability and inflammatory response but the acceleration effect could be rescued by GAS5-overexpression. Meanwhile, miR-18a-5p was recognized as the target gene of GAS5. Conclusion This study indicated that the expression level of GAS5 in the serum of TB patients was decreased, while in the cells infected with MTB, the down-regulated GAS5 might develop a role in facilitating the cell vitality and the inflammatory response by adsorbing miR-18a-5p in the form of molecular sponge.

Background/aim Serum-derived macrophage activating factor (serum-MAF) is expected to have adjuvant effects through rapid phagocytic activation, which depends on F-actin accumulation in multi-layered membrane ruffles induced within 5 min after serum-MAF addition. This study aimed to elucidate the importance of annexin A2, which is a multifunctional Ca2+-binding protein related to cytoskeletal membrane dynamics, in serum-MAF signalling. Materials and methods Annexin A2 and F-actin localizations were analyzed via immunostaining and confocal microscopy. Using EGTA as chelator, the role of Ca2+ in serum-MAF signalling was examined. Results Annexin A2 was found to translocate from the cytosol to the cell cortex within 30 s of serum-MAF stimulation. Ca2+ chelation inhibited the translocation of annexin A2, frill-like structure formation, and phagocytic activation by serum-MAF. Conclusion Annexin A2 and Ca2+ were responsible for the rapid phagocytic activation by serum-MAF. This study provides an understanding of phagocytic activation in macrophages, which could be beneficial for cancer immunotherapy.

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties.

Systemic inflammation as manifested in sepsis is an excessive, life-threatening inflammatory response to severe bacterial or viral infection or extensive injury. It is also a thrombo-inflammatory condition associated with vascular leakage/hemorrhage and thrombosis that is not effectively treated by current anti-inflammatory or anti-thrombotic drugs. Here, we show that MB2mP6 peptide nanoparticles, targeting the Gα13-mediated integrin “outside-in” signaling in leukocytes and platelets, inhibited both inflammation and thrombosis without causing hemorrhage/vascular leakage. MB2mP6 improved mouse survival when infused immediately or hours after onset of severe sepsis. Furthermore, platelet Gα13 knockout inhibited septic thrombosis whereas leukocyte Gα13 knockout diminished septic inflammation, each moderately improving survival. Dual platelet/leukocyte Gα13 knockout inhibited septic thrombosis and inflammation, further improving survival similar to MB2mP6. These results demonstrate that inflammation and thrombosis independently contribute to poor outcomes and exacerbate each other in systemic inflammation, and reveal a concept of dual anti-inflammatory/anti-thrombotic therapy without exacerbating vascular leakage., Bacterial or viral infection can lead to lethal systemic inflammation and thrombosis. Here, the authors show that inhibiting integrin outside-in signaling in leukocytes and platelets alleviates inflammation/clotting and improved survival in septic mice.

Inflammation is a fundamental pathophysiological process which occurs in the course of several diseases. The present work describes the capacity of phytoprostanes (PhytoPs) and phytofurans (PhytoFs) (plant oxylipins), present in plant-based foods, to modulate inflammatory processes mediated by prostaglandins (PGs, human oxylipins) in lipopolysaccharide (LPS)-stimulated THP-1 monocytic cells, through a panel of 21 PGs and PG's metabolites, analyzed by UHPLC-QqQ-ESI-MS/MS. Also, the assessment of the cytotoxicity of PhytoPs and PhytoFs on THP-1 cells evidenced percentages of cell viability higher than 90% when treated with up to 100 μM. Accordingly, 50 μM of the individual PhytoPs and PhytoFs 9-F-PhytoP, 9-epi-9-F-PhytoP, ent-16-F-PhytoP, ent-16-epi-16-F-PhytoP, ent-9-D-PhytoP, 16-B-PhytoP, 9-L-PhytoP, ent-16(RS)-9-epi-ST-Δ-10-PhytoF, ent-9(RS)-12-epi-ST-Δ-13-PhytoF, and ent-16(RS)-13-epi-ST-Δ-9-PhytoF were evaluated on their capacity to modulate the expression of inflammatory markers. The results obtained demonstrated the presence of 7 metabolites (15-keto-PGF, PGF, 11β-PGF, PGE, PGD, PGDM, and PGF) in THP-1 monocytic cells, which expression was significantly modulated when exposed to LPS. The evaluation of the capacity of the individual PhytoPs and PhytoFs to revert the modification of the quantitative profile of PGs induced by LPS revealed the anti-inflammatory ability of 9-F-PhytoP, ent-9-D-PhytoP, 16-B-PhytoP, 9-L-PhytoP, and ent-9(RS)-12-epi-ST-Δ-13-PhytoF, as evidenced by their capacity to prevent the up-regulation of 15-keto-PGF, PGF, PGE, PGF, PGDM, and PGD induced by LPS. These results indicated that specific plant oxylipins can protect against inflammatory events, encouraging further investigations using plant-based foods rich in these oxylipins or enriched extracts, to identify specific bioactivities of the diverse individual molecules, which can be useful for nutrition and health in the frame of well-defined pathophysiological processes., This work was partially funded by the “Fundación Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15, and the Spanish project AGL2017-83386-R from the Spanish Ministry of Science, Innovation, and Universities. This work is included in the framework of the collaboration between the Spanish Research Council (CEBAS-CSIC) and CNRS-University of Montpellier, as “Projets Internationaux de Cooperation Scientifique” (PICS-2015-261141)

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS, a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

Recent studies have provided emerging evidence of the critical involvement of microRNAs in host immune defence against bacterial infection and that likewise the expression of the miRNAs is profoundly impacted by a variety of pathogens to subvert the immune response. Here, we report the role of hsa-let-7a miRNA in response to Staphylococcus epidermidis Small Colony Variants infection. We also assessed whether the expression levels of inflammatory cytokines associated with the hsa-let-7a are manipulated by the pathogen and the effect of the IFN-γ priming on the expression of hsa-let-7a and the fate of SCVs/WTs in infected macrophages. A striking observation was the downregulation of the let-7a miRNA upon challenge of the THP-1 activated cells with the SCV isolates while no significant changes in expression were noticed after the infection of macrophages with their WT counterparts. Staphylococcus epidermidis WT and SCV strains were found to invade and survive in macrophages. A significant reduction in bacterial load for both phenotypes was observed in macrophages treated with let-7a mimic compared to untreated ones. Survival of WTs was augmented in cells treated with the inhibitor in 4 out of 5 strains as compared to the number of bacteria recovered from non-transfected cells. At the same time, let-7a inhibitor did not influence on the survival of SCVs in macrophages as their number was comparable to number recovered from non-transfected cells. When the ratio of both let-7a cytokine targets was compared, anti-inflammatory IL-10 cytokine was induced by SCVs predominantly, while the macrophage challenge with WTs was characterized by the inflammatory cytokine profile with high IL-6 and low IL-10 production. Moreover, the balance between pro-inflammatory and anti-inflammatory cytokines has been expectedly retrieved when macrophages were transfected with let-7a mimic before infection with WT or SCV strains. The results also show that IFN-γ likely regulates the macrophage environment contributing to the inflammatory response and elimination of bacteria from intracellular milieu by augmenting the synthesis of pro-inflammatory cytokines and supressing the anti-inflammatory IL-10. Our work has shown that SCVs have the potential to regulate the let-7a miRNA to balance the pro-inflammatory IL-6 with anti-inflammatory IL-10 and this mechanism is one of the ways in a complex regulatory network adopted by SCVs to promote their survival.

Sangiliyandi Gurunathan, Min-Hee Kang, Muniyandi Jeyaraj, Jin-Hoi Kim Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, KoreaCorrespondence: Jin-Hoi KimDepartment of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, 05029, KoreaTel +82 2 450 3687Fax +82 2 544 4645Email jhkim541@konkuk.ac.krBackground: Exosomes are endosome-derived nano-sized vesicles that have emerged as important mediators of intercellular communication and play significant roles in various diseases. However, their applications are rigorously restricted by the limited secretion competence of cells. Therefore, strategies to enhance the production and functions of exosomes are warranted. Studies have shown that nanomaterials can significantly enhance the effects of cells and exosomes in intercellular communication; however, how palladium nanoparticles (PdNPs) enhance exosome release in human leukemia monocytic cells (THP-1) remains unclear. Therefore, this study aimed to address the effect of PdNPs on exosome biogenesis and release in THP-1 cells.Methods: Exosomes were isolated by ultracentrifugation and ExoQuickTM and characterized by dynamic light scattering, nanoparticle tracking analysis system, scanning electron microscopy, transmission electron microscopy, EXOCETTM assay, and fluorescence polarization. The expression levels of exosome markers were analyzed via quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.Results: PdNP treatment enhanced the biogenesis and release of exosomes by inducing oxidative stress, endoplasmic reticulum stress, apoptosis, and immunomodulation. The exosomes were spherical in shape and had an average diameter of 50– 80 nm. Exosome production was confirmed via total protein concentration, exosome counts, acetylcholinesterase activity, and neutral sphingomyelinase activity. The expression levels of TSG101, CD9, CD63, and CD81 were significantly higher in PdNP-treated cells than in control cells. Further, cytokine and chemokine levels were significantly higher in exosomes isolated from PdNP-treated THP-1 cells than in those isolated from control cells. THP-1 cells pre-treated with N-acetylcysteine or GW4869 showed significant decreases in PdNP-induced exosome biogenesis and release.Conclusion: To our knowledge, this is the first study showing that PdNPs stimulate exosome biogenesis and release and simultaneously increase the levels of cytokines and chemokines by modulating various physiological processes. Our findings suggest a reasonable approach to improve the production of exosomes for various therapeutic applications.Keywords: exosomes, biogenesis, human leukemia monocytic cells, palladium nanoparticles, oxidative stress, endoplasmic reticulum stress

Poly(lactide‐co‐glycolide) (PLGA) shows great potentials in biomedical applications, in particular with the field of biodegradable implants and control release technologies. However, there are few systematic and detailed studies on the influence of PLGA degradation behavior on the immunogenicity. In this study, in order to develop a method for dynamically assessing the immunological response of PLGA throughout the implantation process, PLGA particles are fabricated using an o/w single‐emulsion method. The physicochemical characterizations of the prepared PLGA particles during in vitro hydrolytic degradation are investigated. Then, a series of immunological effects triggered by PLGA by‐products formed with degradation process are evaluated, including cell viability, apoptosis, polarization and inflammatory reaction. THP‐1 human cell line is set as in vitro cell model. Our results show that PLGA degradation‐induced acid environment decreases cell viability and increases cell apoptosis, which is a potential factor affecting cell function. In particular, the macrophages exhibit up‐regulations in both M1 subtype related surface markers and pro‐inflammatory cytokines with the degradation process of PLGA, which indicates the degradation products of PLGA can convert macrophages to the pro‐inflammatory (M1) polarization state. All these findings provide the mechanism of PLGA‐induced inflammation and lay the foundation for the design of next‐generation PLGA‐based biomaterials endowed with immunomodulatory functions.

Objectives: Blau syndrome is a distinct class of autoinflammatory syndrome presenting with early-onset systemic granulomatosis. Blau syndrome-causing NOD2 mutations located in the central nucleotide-oligomerization domain induce ligand-independent basal NF-κB activation in an in vitro reporter assay. However, the precise role of this signaling on granuloma formation has not yet been clarified. Methods: Blau syndrome-causing NOD2 mutations were introduced into human monocytic THP-1 cells, and their morphological and molecular changes from parental cells were analyzed. Identified molecules with altered expression were examined in the patient’s lesional skin by immunostaining. Results: Although the production of proinflammatory cytokines was not altered without stimulation, mutant NOD2-expressing THP-1 cells attached persistently to the culture plate after stimulation with phorbol myristate acetate. Sustained surface ICAM-1 expression was observed in association with this phenomenon, but neither persistent ICAM-1 mRNA expression nor impaired ADAM17 mRNA expression was revealed. However, the transient induction of PDGF-B mRNA expression was specifically observed in stimulated THP-1 derivatives. In the granulomatous skin lesion of a Blau syndrome patient, ICAM-1 and PDGF-B were positively immunostained in NOD2-expressing giant cells. Conclusions: Sustained surface ICAM-1 expression and transient PDGF-B production by newly differentiating macrophages harboring mutant NOD2 might play a role in granuloma formation in Blau syndrome.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Summary Flow cytometry is a valuable method for analyzing protein expressions at the single cell level but can be difficult to apply to large numbers of samples. This protocol provides instructions to perform a high-throughput small molecule screen using flow cytometry analysis of THP-1 cells, a human monocytic leukemia cell line. We describe a methodology for identifying compounds that regulate PD-L1 surface expression in IFN-γ-stimulated cells, which has been successfully used to screen a collection of ∼200,000 compounds. For complete details on the use and execution of this protocol, please refer to Zavareh et al. (2020)., Graphical Abstract, Highlights • Protocol for high-throughput screening of compounds using flow cytometry • Designed for quantification of cell surface expression of proteins in THP-1 cells • This protocol has been used to identify modulators of PD-L1 expression, Flow cytometry is a valuable method for analyzing protein expressions at the single cell level but can be difficult to apply to large numbers of samples. This protocol provides instructions to perform a high-throughput small molecule screen using flow cytometry analysis of THP-1 cells, a human monocytic leukemia cell line. We describe a methodology for identifying compounds that regulate PD-L1 surface expression in IFN-γ-stimulated cells, which has been successfully used to screen a collection of ∼200,000 compounds.

Background Tinospora cordifolia (Willd).Miers is a perennial climbing medicinal shrub that has been traditionally used for the treatment of chronic inflammatory ailments. Our previous pre- clinical studies on anti-inflammatory effects, proved that the chloroform extract of T. cordifolia (CETC) suppressed the LPS induced up-regulation of pro-inflammatory biomarkers, hence, further follow up study was carried out to evaluate whether CETC can exhibit a protective effect against LPS induced lethal endotoxemia in vivo and also to analyze the impact of CETC pre-treatment on the secretion of pro-inflammatory cytokines in vitro by THP-1 cells. Methods To corroborate our previous preclinical studies on inflammation, we investigated the mechanism of the anti-inflammatory effect of T. cordifolia on THP-cells which were pre-incubated with CETC (30 min) and stimulated subsequently with LPS (1 μg/ml) for 20 h. Levels as well as gene expressions of various cytokines were compared with that of LPS alone incubated cells. Alongside, in vivo oral anti-inflammatory efficacy against LPS induced endotoxemia study was effectuated, wherein rats were administered with CETC 48, 24, 12 and 1 h prior to the injection of LPS and the survival of rats were monitored upto 10 days. Cytokine levels were quantified by ELISA. Nitrite levels were measured using Griess reagent. Expression of pro-inflammatory proteins was inspected in rat tissues by histochemical and immuno -histochemical examinations. Results CETC was able to down-regulate the up-regulation of pro-inflammatory biomarkers in THP-1 macrophages though blockade of NF-κB nuclear translocation and could improve the survival rate during endotoxemic episodes with a marked suppression of the tissue expression of pro-inflammatory proteins. Conclusion These findings concomitantly reveal the anti-inflammatory mechanism of CETC and support us to move forward for the development of drugs against disorders resulting from deregulated immune reactions.

Hydrogen sulfide (H2S) exerts an anti-atherosclerotic effect and decreases foam cell formation. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a key factor involved in foam cell formation. However, the association between H2S and Lp-PLA2 expression levels with respect to foam cell formation has not yet been elucidated. The present study investigated whether H2S can affect foam cell formation and potential signalling pathways via regulation of the expression and activity of Lp-PLA2. Using human monocytic THP-1 cells as a model system, it was observed that oxidized low-density lipoprotein (ox-LDL) not only upregulates the expression level and activity of Lp-PLA2, it also downregulates the expression level and activity of Cystathionine γ lyase. Exogenous supplementation of H2S decreased the expression and activity of Lp-PLA2 induced by ox-LDL. Moreover, ox-LDL induced the expression level and activity of Lp-PLA2 via activation of the p38MAPK signalling pathway. H2S blocked the expression levels and activity of Lp-PLA2 induced by ox-LDL via inhibition of the p38MAPK signalling pathway. Furthermore, H2S inhibited Lp-PLA2 activity by blocking the p38MAPK signaling pathway and significantly decreased lipid accumulation in ox-LDL-induced macrophages, as detected by Oil Red O staining. The results of the present study indicated that H2S inhibited ox-LDL-induced Lp-PLA2 expression levels and activity by blocking the p38MAPK signalling pathway, thereby improving foam cell formation. These findings may provide novel insights into the role of H2S intervention in the progression of atherosclerosis.

MicroRNAs are widely considered to be involved in the pathogenesis of atherosclerosis. Whereas the importance of miR-30a-5p as a tumor growth-promoting factor has been extensively studied, the relationship between this particular microRNA and atherosclerosis remains to be clarified. In this study, the role of miR-30a-5p in the formation of foam cells from THP-1-derived macrophages was investigated. It was found that miR-30a-5p could robustly regulate the pathological process of atherosclerosis by inhibiting autophagy and increasing the accumulation of lipids, the expression of inflammatory factors, and the apoptosis of macrophages. These results provide guidance for future assessments of the progression of atherosclerosis and for the development of intervention targets for the treatment of this disease.

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

HIGHLIGHTS L. duriusculum n-BuOH extract reduces inflammatory responses both in vitro and in vivo. L. duriusculum n-BuOH extract inhibits NF-κB-dependent transcriptional responses. L. duriusculum n-BuOH extract decreases the expression of TNF-α and IL-6 genes.

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-β1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells., This research was funded by EU FEDER funds, Interreg España-Portugal Programme, under the framework of the Project: ref. 0377_IBERPHENOL_6_E; by Xunta de Galicia funds, Spain (CITACA Strategic Partnership, Reference: ED431E 2018/07) and also supported by UIDB/50006/2020 and PTDC/SAU-NUT/30448/2017 with funding from FCT/MCTES through national funds. I. F. acknowledge her Fundação para a Ciência e Tecnologia (FCT) research contract (SFRH/BPD/86173/2012).

Background The genomes of HIV-2 and some SIV strains contain the accessory gene vpx, which carries out several functions during infection, including the downregulation of SAMHD1. Vpx is also commonly used in experiments to increase HIV-1 infection efficiency in myeloid cells, particularly in studies that investigate the activation of antiviral pathways. However, the potential effects of Vpx on cellular innate immune signaling is not completely understood. We investigated whether and how Vpx affects ISG responses in monocytic cell lines and MDMs during HIV-1 infection. Results HIV-1 infection at excessively high virus doses can induce ISG activation, although at the expense of high levels of cell death. At equal infection levels, the ISG response is potentiated by the presence of Vpx and requires the initiation of reverse transcription. The interaction of Vpx with the DCAF1 adaptor protein is important for the enhanced response, implicating Vpx-mediated degradation of a host factor. Cells lacking SAMHD1 show similarly augmented responses, suggesting an effect that is independent of SAMHD1 degradation. Overcoming SAMHD1 restriction in MDMs to reach equal infection levels with viruses containing and lacking Vpx reveals a novel function of Vpx in elevating innate immune responses. Conclusions Vpx likely has as yet undefined roles in infected cells. Our results demonstrate that Vpx enhances ISG responses in myeloid cell lines and primary cells independently of its ability to degrade SAMHD1. These findings have implications for innate immunity studies in myeloid cells that use Vpx delivery with HIV-1 infection.

Gout is a chronic inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystal deposits in joints of lower limbs. Phagocytic uptake of MSU crystals by joint-resident macrophages and recruited circulating monocytes results in IL-1β expression and production. Current acute gout treatments have serious toxicities and suffer suboptimal clinical outcomes. Protein phosphatase 2A (PP2A) plays an important role in regulating signaling pathways relevant to inflammation. We hypothesized that innate immune danger signals, e.g., lipopolysaccharide (LPS) and soluble uric acid (sUA), prime human monocytes toward MSU crystal phagocytosis and that increased IL-1β production mediated by a reduction in PP2A activity and restoring PP2A activity exerts an anti-inflammatory effect in this setting. Priming monocytes with LPS + sUA increased cytosolic pro-IL-1β and mature IL-1β and enhanced MSU crystal phagocytosis and its downstream IL-1β expression (P < 0.001). A combination of LPS + sUA priming and MSU crystals reduced PP2A activity in monocytes by 60% (P = 0.013). PP2A catalytic subunit gene knockdown reduced PP2A activity and exacerbated MSU crystal-induced IL-1β expression and secretion (P < 0.0001). Fingolimod (FTY720) and its active metabolite, fingolimod phosphate (FTY720-P), were evaluated for their ability to activate PP2A in human monocytes over 24 hours. FTY720 and FTY720-P activated PP2A to a similar extent, and maximal enzyme activity occurred at 24 hours for FTY720 and at 6 hours for FTY720-P. FTY720-P (2.5 μM) reduced pro-IL-1β production and IL-1β secretion in primed and MSU crystal-stimulated monocytes (P < 0.0001) without changing the magnitude of crystal phagocytosis. We conclude that PP2A is a promising new target in acute gout. SIGNIFICANCE STATEMENT: The activity of protein phosphatase 2A (PP2A) is implicated in the enhanced expression and production of IL-1β by human monocytes in response to priming with soluble uric acid and lipopolysaccharide and phagocytosis of monosodium urate monohydrate (MSU) crystals. Fingolimod phosphate activates PP2A in human monocytes and reduces cytosolic pro-IL-1β content and its conversion to biologically active IL-1β in human monocytes exposed to MSU crystals.

Background: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and a catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the mono-, di-, and tri-methylation of histone H3 at Lys 27 (H3K27me3) to facilitate chromatin remodeling and gene silencing functions. The overexpression of EZH2 is associated with high levels of H3K27me3 in various types of cancers. Previous reports showed a significant association of EZH2 aberrations in pediatric cancers such as soft tissue sarcomas and glioblastoma. Recent reports in human subjects and animal models have also suggested a central role of EZH2 in the induction and progression of acute myeloid leukemia. Methods: In this study, we aimed to investigate the molecular status of EZH in cell lines derived from distinct pediatric leukemia to assess the efficacy of targeting EZH2 to suppress cancer cell survival and proliferation. Cell cytotoxicity and the half maximal inhibitory concentration (IC50) were measured by Alamar blue cytotoxicity assay. The molecular status of EZH2 was profiled by DNA sequencing and western blot analysis of EZH2 protein levels in THP-1, SEM, and MV4:11. In addition, Methylation of histone H3 was evaluated by immunoblotting and immunostaining analyses. Results: Our results showed that EZH2 protein is overexpressed in the pediatric monocytic cell line THP-1 , but not in other leukemia derived cell lines MV4;11 and SEM. Screening a panel of methyltransferase inhibitors revealed that three inhibitors; GSK126, UNC1999 and EPZ-5687 are the most potent inhibitors that suppressed EZH2 activity selectively on lysine 27 which resulted in increased apoptosis and inhibition of AKT and ERK protein phosphorylation in THP-1 cells. Our data demonstrated a significant increase in apoptosis in cells treated with drug combination (EZH2i and selinexor) compared to EZH2i inhibitors alone. Conclusions: Taken together, our data provide initial evidence that targeting EZH2 is a promising therapeutic strategy for the treatment of subtypes of pediatric AML. Also, combining EZH2 inhibitors with selinexor may increase the treatment efficacy in these patients. Findings from this study indicate further evaluation and consideration of these compounds for early phase clinical studies in selected pediatric malignancies in the future.

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.

Allergen recognition and processing by antigen presenting cells is essential for the sensitization step of food allergy. Macrophages and dendritic cells are both phagocytic antigen presenting cells and play important roles in innate immune responses and signaling between the innate and adaptive immune system. To obtain a model system with a homogeneous genetic background, we derived macrophages and dendritic cells from THP-1 monocytes. The difference between macrophages and dendritic cells was clearly shown by differences in their transcription response (microarray) and protein expression levels. Their resemblance to primary cells was analyzed by comparison to properties as described in literature. The uptake of β-lactoglobulin after wet-heating (60°C in solution) by THP-1 derived macrophages was earlier reported to be significantly increased. To analyse the subsequent immune response, we incubated THP-1 derived macrophages and dendritic cells with native and differently processed β-lactoglobulin and determined the transcription and cytokine expression levels of the cells. A stronger transcriptional response was found in macrophages than in dendritic cells, while severely structurally modified β-lactoglobulin induced a more limited transcriptional response, especially when compared to native and limitedly modified β-lactoglobulin. These results show that processing is relevant for the transcriptional response toward β-lactoglobulin of innate immune cells.

Cutibacterium acnes (formerly Propionibacterium acnes) is one of the major bacterial species responsible for acne vulgaris. Numerous bioactive compounds from Momordica charantia Linn. var. abbreviata Ser. have been isolated and examined for many years. In this study, we evaluated the suppressive effect of two cucurbitane-type triterpenoids, 5&beta, 19-epoxycucurbita-6,23-dien-3&beta, 19,25-triol (Kuguacin R, KR) and 3&beta, 7&beta, 25-trihydroxycucurbita-5,23-dien-19-al (TCD) on live C. acnes-stimulated in vitro and in vivo inflammatory responses. Using human THP-1 monocytes, KR or TCD suppressed C. acnes-induced production of interleukin (IL)-1&beta, IL-6 and IL-8 at least above 56% or 45%, as well as gene expression of these three pro-inflammatory cytokines. However, a significantly strong inhibitory effect on production and expression of tumor necrosis factor (TNF)-&alpha, was not observed. Both cucurbitanes inhibited C. acnes-induced activation of the myeloid differentiation primary response 88 (MyD88) (up to 62%) and mitogen-activated protein kinases (MAPK) (at least 36%). Furthermore, TCD suppressed the expression of pro-caspase-1 and cleaved caspase-1 (p10). In a separate study, KR or TCD decreased C. acnes-stimulated mouse ear edema by ear thickness (20% or 14%), and reduced IL-1&beta, expressing leukocytes and neutrophils in mouse ears. We demonstrated that KR and TCD are potential anti-inflammatory agents for modulating C. acnes-induced inflammation in vitro and in vivo.

Amaç: Makrofajlar doğal immün cevabı başlatan ve patojenle ilk temasta bulunan hücreler olarak kabul edilmektedir. Hem hücreler arası ilişkiler hem de inflamatuar mediyatörlerin salınımı yoluyla doğal immün ve inflamatuar yanıtta etkin rol oynamaktadırlar. İnsan THP-1 lösemi monositik hücreleri, makrofajların in vitro olarak fonksiyonlarını, mekanizmalarını ve sinyal yollarını araştırmak için en çok kullanılan hücre dizisidir. Lipopolisakkarit makrofaj farklılaşmasını başlatmak için yaygın olarak kullanılan uyarıcılar arasında yer almaktadır. Ferulik asidin, indüklenebilir nitrik oksit sentaz, kaspazlar ve siklooksijenaz (COX)-2 dahil olmak üzere proinflamatuar sitokinlerin ekspresyonunu ve/veya aktivitesini inhibe ettiği bilinmektedir. Bu çalışmada, lipopolisakkarit ile indüklenen THP-1 monosit hücrelerinde Ferulik asidin sitokin (COX-1, IL-lα, IL-1β, TNF-α, IL-6, IL-10, NF-kβ ve IFN-y) düzeyleri üzerindeki etkisini araştırmayı amaçladık.Gereç ve Yöntem: TNF alfa seviyeleri farklı konsantrasyonlarda ve zamanlarda LPS eklenerek ölçüldü ve en uygun konsantrasyon ve süre belirlendi. COX-1, IL-lα, IL-lβ, TNF-α, IL-6, IL-10, NF-kβ ve IFN-y'nin sitokin miktarları, inkübasyon sürelerinin sonunda toplanan süpernatanlarda ELISA ile ölçüldü.Bulgular: Ferulik asidin, NF-kβ inhibisyonu ile lipopolisakkarit ile indüklenen THP-1 hücrelerinde artan TNF-α, IL-lα ve IL-lβ ekspresyonunu inhibe ettiğini bulduk.Sonuç: Ferulik asidin LPS ile indüklenen THP-1 hücrelerinde sitokin salınımı üzerindeki etkisinin gösterilmesi, aşırı inflamatuar yanıtın tedavisinde ve oto-immün hastalıklara karşı korunmada etkili olabileceğini düşündürdü., Aim: Macrophages are accepted as cells that initially contact with the pathogens and initiate the innate immune response. They play effective roles in innate immune and inflammatory responses by intercellular relations and inflammatory mediator secretion. Human THP-1 leukemia monocytic cells are frequently used for the in vitro determination of the signal pathways, and the functions of macrophages. Lipopolysaccharide is commonly used to induce macrophage differentiation of monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear.Ferulic acid (FA) is known to inhibit the expression and / or activity of proinflammatory cytokines, including inducible nitric oxide synthase, caspases, and cyclooxygenase (COX)-2. In this study, we aimed to investigate the effect of Ferulic acid on cytokine (COX-1, IL-1α, IL-1β, TNF-α, IL-6, IL-10, NF-kβ and IFN-γ) levels in lipopolysaccharide induced THP-1 monocyte cells.Materials and Methods: TNF alpha levels were measured by performing applications at different concentrations and times, and the most appropriate concentration and duration were determined. Cytokine levels of COX-1, IL-1α, IL-1β, TNF-α, IL-6, IL-10, NF-kβ and IFN-γ were measured by ELISA in supernatants collected at the end of incubation times from wells.Results: We have found that ferulic acid inhibits the expression of TNF-α, IL-1α and IL-1β in LPS-induced macrophages by inhibition of NF-kB.Conclusion: It has been found that the effect of ferulic acid on cytokine release in LPS-induced THP-1 cells may be effective in the treatment of excessive inflammatory response and protection against auto-immune diseases.

Presently, the attention given to natural substances to counteract damage produced by oxidative stress (OS) has risen sharply. In this scenario, hydroxytyrosol (HT) derivatives, formed as a result of HT conjugation with fatty acids (FAs) (lipophenols), have been recently described in foodstuffs such as extra virgin olive oil, as being powerful bioactive compounds with a higher activity than the unesterified phenolic compound. The present work describes the capacity of HT lipophenols to act on the course of OS and secondary inflammatory processes, based on their capacity to modulate the isoprostanoid profile induced by HO in THP-1 monocytic cells. A UHPLC-QqQ-ESI-MS/MS-based lipidomics workflow was applied over a range of 37 human oxylipins. The main outcomes retrieved suggest both HT and HT-lipophenols as regulators of the cellular redox balance, acting as pro-oxidants in vitro, which is highly dependent on the experimental conditions. Our outcomes suggest the anti-inflammatory potential of both HT and HT-lipophenols, where the type of the FAs on the HT core appears to be critical for defining the bioactivity of lipophenols, highlighting that a lipidomic approach, with the simultaneous analysis of multiple oxylipins, is critical for the understanding of the bioactivity of lipophenols on isoprostanoid generation and hence, on pathophysiological processes., This work was partially funded by the “Fundación Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15 and the Spanish project AGL2017-83386-R-AEI/FEDER, UE from the Spanish Ministry of Science, Innovation, and Universities. SM was supported by a Postdoctoral Contract (Saavedra-Fajardo 21078/SF/19) from the Agency for Science and Technology of the Región de Murcia – “Fundación Séneca” (Spain)

We investigated the fundamental function of the lung cancer tumour microenvironment. Proteasome inhibition has emerged as a clinically successful anti-cancer therapeutic strategy, with the antineoplastic medication bortezomib showing high efficacy against multiple cancers. Tumour-associated macrophages (TAMs), which migrate to tumour stroma, are known to promote cell proliferation, apoptosis, and metastasis within the lung cancer microenvironment. However, the specific interaction of macrophages, lung cancer cells, and bortezomib are still unclear. Most in vitro cell cultures are traditionally grown in a two-dimensional (2D) culture system, although 3D cultures have demonstrated greater success in terms of drug response, gene expression and viability. Therefore, to elucidate the role of macrophages, we aimed to establish a co-culture model in both 2D and 3D cultures using A549 human lung cancer cells and THP-1 derived macrophages. Apoptotic effects were analysed using the Annexin V-PI method. Since NF-kB and TNF-alpha play important roles in the anti-proliferation and apoptosis pathways, the levels of NF-kB and TNF-alpha mRNA expression were measured. As a result, bortezomib significantly increased cell apoptosis in cells co-cultured with M0 macrophages. IL-1 beta cytokine levels also increased, and NF-kB mRNA expression levels decreased. Understanding the mechanisms that modulate the tumour microenvironment may facilitate the development of novel anticancer therapies. The utilisation of TAMs may improve the successful treatment of lung cancer and warrants further study.

Xanthone derivatives have shown promising antitumor properties, and 1-carbaldehyde-3,4-dimethoxyxanthone (1) has recently emerged as a potent tumor cell growth inhibitor. In this study, its effect was evaluated (MTT viability assay) against a new panel of cancer cells, namely cervical cancer (HeLa), androgen-sensitive (LNCaP) and androgen-independent (PC-3) prostate cancer, and nonsolid tumor derived cancer (Jurkat) cell lines. The effect of xanthone 1 on macrophage functions was also evaluated. The effect of xanthone 1-conditioned THP-1 human macrophage supernatants on the metabolic viability of cervical and prostate cancer cell lines was determined along with its interference with cytokine expression characteristic of M1 profile (IL-1 ≤ β; TNF-α) or M2 profile (IL-10; TGF-β) (PCR and ELISA). Nitric oxide (NO) production by murine RAW264.7 macrophages was quantified by Griess reaction. Xanthone 1 (20 μM) strongly inhibited the metabolic activity of the cell lines and was significantly more active against prostate cell lines compared to HeLa (p < 0.05). Jurkat was the cell most sensitive to the effect of xanthone 1. Compound 1-conditioned IL-4-stimulated THP-1 macrophage supernatants significantly (p < 0.05) inhibited the metabolic activity of HeLa, LNCaP, and PC-3. Xanthone 1 did not significantly affect the expression of cytokines by THP-1 macrophages. The inhibiting effect of compound 1 observed on the production of NO by RAW 264.7 macrophages was moderate. In conclusion, 1-carbaldehyde-3,4-dimethoxyxanthone (1) decreases the metabolic activity of cancer cells and seems to be able to modulate macrophage functions.

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150&ndash, 450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1&beta, -6, -8, and TNF-&alpha, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p &lt, 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/&mu, L for IL1R1 and COX-2 (p &lt, 0.01) and 300 ng/&mu, L for NF-&kappa, B (p &lt, 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Atherosclerosis is a long-term disease process of the vascular system that is characterized by the formation of atherosclerotic plaques, which are inflammatory regions on medium and large-sized arteries. There are many factors contributing to plaque formation, such as changes in shear stress levels, rupture of endothelial cells, accumulation of lipids, and recruitment of leukocytes. Shear stress is one of the main factors that regulates the homeostasis of the circulatory system; therefore, sudden and chronic changes in shear stress may cause severe pathological conditions. In this study, microfluidic channels with cavitations were designed to mimic the shape of the atherosclerotic blood vessel, where the shear stress and pressure difference depend on design of the microchannels. Changes in the inflammatory-related molecules ICAM-1 and IL-8 were investigated in THP-1 cells in response to applied shear stresses in an continuous cycling system through microfluidic channels with periodic cavitations. ICAM-1 mRNA expression and IL-8 release were analyzed by qRT-PCR and ELISA, respectively. Additionally, the adhesion behavior of sheared THP-1 cells to endothelial cells was examined by fluorescence microscopy. The results showed that 15 Pa shear stress significantly increases expression of ICAM-1 gene and IL-8 release in THP-1 cells, whereas it decreases the adhesion between THP-1 cells and endothelial cells.

Pyrus pyrifolia Nakai (P. pyrifolia) has been traditionally used in East Asia to treat diseases such as phlegm, cough, hangover, and fever. However, there is no investigation that evaluates the biological activities of the leaves of P. pyrifolia. This study aims at describing the anti-inflammatory effects of PP, a bioactive fraction from the leaves of P. pyrifolia, in lipopolysaccharide (LPS)-stimulated THP-1 cells. Initially, PP decreased the protein and RNA expression of TNF-α, MCP-1, IL-8, and IL-6 induced by LPS. Moreover, PP attenuated the phosphorylation of p38, JNK, and ERK. In addition, after stimulation with LPS, the degradation of IκB-α was suppressed by PP, and the phosphorylation of IκB-α and p65 was suppressed by PP. Additionally, PP increased HO-1, which controls the production of inflammatory molecules, by activating Nrf2. These results indicated that PP could be used as an anti-inflammatory drug to promote wellness.

Background Competitive endogenous RNA (ceRNA) reveals new mechanisms for interactions between RNAs, which have been considered to play a significant role in pathogen-host innate immune response. However, knowledge of ceRNA regulatory networks in Talaromyces marneffei (TM)-macrophages is still limited. Methods Next-generation sequencing technology (NGS) was used to obtain mRNA, miRNA and lncRNA expression profiles in TM-infected macrophages. The R package DESeq2 was used to identify differentially expressed lncRNA, miRNA and mRNA. The R package GOseq was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and the ceRNA network of lncRNA–miRNA–mRNA interaction was constructed in Cytoscape. Similarly, functional enrichment analysis on mRNA in the ceRNA network. Finally, two mRNAs and four lncRNAs in the ceRNA network were randomly selected to verify the expression using qRT-PCR. Results In total, 119 lncRNAs, 28 miRNAs and 208 mRNAs were identified as differentially expressed RNAs in TM-infected macrophages. The constructed ceRNA network contains 38 lncRNAs, 10 miRNAs and 45 mRNAs. GO and KEGG analysis of mRNA in the ceRNA network indicated that activated pathways in TM-infected macrophages were related to immunity, inflammation and metabolism. The quantitative validation of the expression of four randomly selected differentially expressed lncRNAs, AC006252.1, AC090197.1, IL6R-AS1, LINC02009 and two mRNAs, CSF1, NR4A3 showed that the expression levels were consistent with those in the RNA-sequencing. Conclusions The ceRNA network related to immunity, inflammation and metabolism plays an important role in TM-macrophage interaction. This study may provide effective and novel insights for further understanding the underlying mechanism of TM infection.

Background and objectives The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry-based erythrophagocytosis assay was established using phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells and RBCs labelled with PKH26 to assess allo- and autoantibodies to RBCs. Materials and methods THP-1 cells were differentiated into macrophage-like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA-treated THP-1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. Results We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti-D and anti-K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti-Ch) or autoantibodies from patients and pregnant women without haemolysis. Conclusion The flow cytometry-based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Background Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involved in the regulation of cellular growth, proliferation, and more in general, is a common phenomenon in most types of cancers. Thus, natural substances targeting this pathway can be of great therapeutic potential in supporting the treatment of tumor patients. Rhus tripartita (Ucria) Grande is a plant growing in desertic areas which is traditionally used for the treatment of several diseases in Tunisia. In the present work, the biochemical profile of the main compounds present in the plant leaf extract was determined and the anti-leukemic potential of the plant extracts against acute monocytic leukaemia (AML) THP-1 cells was investigated. Methods After HPLC identification of some phenolic compounds present in the plant extract and the quantification of saponin content, the cytotoxic effect of Rhus tripartita extracts on THP-1 cell culture was evaluated using the colorimetric MTT assay for cell viability. THP-1 cells were incubated with medium containing the relative IC50 concentrations of total plant extract, saponin extract and some standard compounds (rutin (R); kaempferol (K); mixture of catechin, epicatechin, and epicatechin-gallate (CEEG); ellagic acid (EA). Finally, qRT-PCR and western blotting analysis were used to evaluate the effect of some flavonoids present in a crude extract of polyphenols and the total extract of saponins on cell survival and apoptosis. Results Analysis of expression level of some gene (PIK3CA, PTEN, AKT1, mTOR, EIF4E, RPS6KB1, and TSC1) involved in the mTOR pathway and the phosphorylation of S6 and AKT proteins allowed to observe that a total Rhus tripartita extract and some of the compounds found in the extract controls THP-1 cell proliferation and apoptosis via regulation of the PI3K-Akt-mTOR signaling pathway. Conclusion Rhus tripartita-induced inhibition of cell cycle and induction of apoptosis may involve the mTOR pathway. Therefore, Rhus tripartita extract may be a useful candidate as a natural anti-cancer drug to support the treatment of AML.

Acute myeloid leukemia (AML) is a highly heterogeneous disease that remains untreatable. MicroRNAs (miRNAs or miRs) play important roles in the pathogenesis of leukemia. miR-21 is highly expressed in multiple types of human cancer and displays oncogenic activities; however, the clinical significance of miR-21 in AML remains unclear. In the present study, it was demonstrated that miR-21 levels were high in patients with AML and in AML cell lines. Further experiments demonstrated that overexpression of miR-21 in Thp-1 human monocytes derived from acute mononuclear leukemia peripheral blood promoted cell proliferation, while downregulation of miR-21-5p, a mature sequence derived from the 5' end of the miR-21 stem-loop precursor (another mature sequence, miR-21-3p, is derived form 3' end of miR-21), inhibited cell proliferation. Specifically, it was observed that overexpression of miR-21 could promote the transition of Thp-1 cells into the S and G2/M phases of the cell cycle, as shown by flow cytometry. Furthermore, inhibition of miR-21-5p arrested cells in the S and G2/M phases. Finally, BCL11B was determined to be a functional target of miR-21-5p by luciferase assays. Our study revealed functional and mechanistic associations between miR-21 and BCL11B in Thp-1 cells, which could serve to guide clinical treatment of AML.

Prostate-derived extracellular vesicles (pEVs) may represent a way to selectively transport cargo molecules from the producing cells to the target cells to allow biological events, both in physiological and pathological circumstances. pEVs cargo participates in the modulation of the inflammatory responses in physiological conditions and during cancer progression. In the present study, we examined the expression levels of miRNA Let-7b, in both precursor and mature forms, in noncancerous and cancerous prostate cell lines, PNT2 and PC3 respectively, and in their extracellular vesicles (EVs) using reverse-transcription quantitative PCR strategies. We showed that miRNA Let-7b was highly expressed in noncancerous cells and strongly decreased in cancerous PC3 cells, while the opposite was observed in the respective EVs, thus supporting the tumor suppressor role of miRNA Let7-b. We also demonstrated that miRNA Let-7b can be transferred to THP-1 cells via EVs, which are known to induce TAM-like polarization. Our results support the view that miRNA Let-7 b, contained in PC3-derived EVs, is associated with the increase in the miRNA Let7-b observed in TAM-like macrophages. Overall, our results indicate that circulating EV-loaded miRNA might be useful biomarkers for prostate cancer progression and might also support a possible use of pEVs as targets for prostate cancer therapy.

Kcnq1 overlapping transcript 1 (kcnq1ot1), an imprinted antisense lncRNA in the kcnq1 locus, acts as a potential contributor to cardiovascular disease, but its role in atherosclerosis remains unknown. The aim of this study was to explore the effects of kcnq1ot1 on atherogenesis and the underlying mechanism. Our results showed that kcnq1ot1 expression was significantly increased in mouse aorta with atherosclerosis and lipid-loaded macrophages. Lentivirus-mediated kcnq1ot1 overexpression markedly increased atherosclerotic plaque area and decreased plasma HDL-C levels and RCT efficiency in apoE−/− mice fed a Western diet. Upregulation of kcnq1ot1 also reduced the expression of miR-452-3p and ABCA1 but increased HDAC3 levels in mouse aorta and THP-1 macrophages. Accordingly, kcnq1ot1 overexpression inhibited cholesterol efflux and promoted lipid accumulation in THP-1 macrophages. In contrast, kcnq1ot1 knockdown protected against atherosclerosis in apoE−/− mice and suppressed lipid accumulation in THP-1 macrophages. Mechanistically, kcnq1ot1 enhanced HDAC3 expression by competitively binding to miR-452-3p, thereby inhibiting ABCA1 expression and subsequent cholesterol efflux. Taken together, these findings suggest that kcnq1ot1 promotes macrophage lipid accumulation and accelerates the development of atherosclerosis through the miR-452-3p/HDAC3/ABCA1 pathway.

Aims This study was done to investigate the anti-inflammatory effects of high molecular weight secretions from Limosilactobacillus reuteri PTCC 1655 probiotic bacteria on lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. Methods and results After culturing the bacterium, the crude cell-free supernatant was fractionated on the basis of molecular weights using ultrafiltration. Also, a heat-killed and sonicated fraction was obtained from the biomass of the bacterial culture. All fractions were used to measure their anti-inflammatory effects on PMA-differentiated THP-1 cells following LPS stimulation by quantifying various cellular markers of inflammation. The results demonstrated that various L. reuteri PTCC 1655-derived fractions, especially the >100 kDa supernatant fraction decreased some of the inflammatory cytokines and mediators, including tumour necrosis factor-α, interleukin-1, nitric oxide, cyclooxygenase-2, matrix metalloproteinase-9 and interleukin-6, which are critical for the pathogenesis of some inflammatory diseases. Conclusion It is concluded that the L. reuteri PTCC 1655-derived high molecular weight fractions significantly reduce inflammation and therefore could be appropriate candidates for future medical studies. Significance and impact of the study Providing new insights about the significance of L. reuteri PTCC 1655-derived extracts and their potential to modulate inflammation.

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy., Anti-androgen therapy inhibits prostate cancer (PC) progression, and is thought to act directly on cancer cells. Here the authors show that androgen receptor is expressed on normal and PC-associated macrophages, and its stimulation alters macrophage secretome to promote migration of cultured PC cell lines.

Trichophyton rubrum is causing an increasing number of invasive infections, especially in immunocompromised and diabetic patients. The fungal invasive infectious process is complex and has not yet been fully elucidated. Therefore, this study aimed to understand the cellular and molecular mechanisms during the interaction of macrophages and T. rubrum. For this purpose, we used a co-culture of previously germinated and heat-inactivated T. rubrum conidia placed in contact with human macrophages cell line THP-1 for 24 h. This interaction led to a higher level of release of interleukins IL-6, IL-2, nuclear factor kappa beta (NF-&kappa, B) and an increase in reactive oxygen species (ROS) production, demonstrating the cellular defense by macrophages against dead fungal elements. Cell viability assays showed that 70% of macrophages remained viable during co-culture. Human microRNA expression is involved in fungal infection and may modulate the immune response. Thus, the macrophage expression profile of microRNAs during co-culture revealed the modulation of 83 microRNAs, with repression of 33 microRNAs and induction of 50 microRNAs. These data were analyzed using bioinformatics analysis programs and the modulation of the expression of some microRNAs was validated by qRT-PCR. In silico analysis showed that the target genes of these microRNAs are related to the inflammatory response, oxidative stress, apoptosis, drug resistance, and cell proliferation.

The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article "A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism" in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol distribution in the three cell lines for future studies, and can used for future method optimization, and for comparison of oxysterol recovery with other analytical techniques.

The pathogenesis of atherosclerosis is complex, evolves, and involves many cell types. Macrophages and vascular smooth muscle cells (VSMCs) are critically involved in atherosclerosis development and progression. Several studies have shown that WNT5A protein is abundantly expressed in human atherosclerotic lesions; however, the mechanism and role of WNT signaling pathway activation is not clearly known. Using THP-1 derived macrophages, and human aortic VSMC cells, we evaluated in vitro how oxidized low-density lipoprotein (oxLDL) and WNT5A signaling interact in these two cell lines. We used western blot, scratch assay, metabolic proliferation assay, as well as immunostaining to analyze the effect of Wnt signaling activation. The results demonstrated that oxLDL, as well as WNT5A (control), induced Disheveled-2 (DVL2) activation and Kif26b degradation, indicating activation of non-canonical Wnt signaling. We found that oxLDL and WNT5A induced FZD5-ROR2 co-localization at the cellular membrane in vitro in THP-1 derived macrophages. Box5 (FZD5 receptor antagonist) inhibited oxLDL-induced DVL2/JNK activation secondary to newly secreted WNT protein from THP-1 derived macrophages. We found that WNT3A (canonical Wnt) and WNT5A showed different roles in this VSMC cell line. These findings indicate that WNT5A is upregulated by oxLDL, promotes foam cell formation, and affects VSMC phenotype and migration in these two cell lines. Also, in these cell lines FZD5 signaling seems to be necessary for lipid accumulation and, through this mechanism, WNT5A could modulate foam cell formation. Thus, our results suggest that WNT5A may contribute to the pathogenesis of vascular disease through modulating macrophage and VSMC behavior.