Human Monocytic Cell Line THP-1 Research Articles

The Effects of the oxLDL/β2GPI/anti-β2GPI Complex on Macrophage Autophagy and its Mechanism.

Previous research has established that the oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex can stimulate macrophages to secrete molecules associated with atherosclerosis (AS), such as monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), and tumor necrosis factor-α (TNF-α). This complex also enhances the uptake of oxLDL, thereby accelerating foam cell formation through the Toll-like receptor-4/nuclear factor kappa B (TLR4/NF-κB) pathway. Given the critical role of macrophage autophagy in the instability of vulnerable atherosclerotic plaques, it is imperative to investigate whether the oxLDL/β2GPI/anti-β2GPI complex influences macrophage autophagy in AS. This study aims to elucidate the effects and underlying mechanisms of the oxLDL/β2GPI/anti-β2GPI complex on macrophage autophagy in AS. Experiments were conducted using murine macrophage RAW264.7 cells and the human monocytic cell line THP-1. Western blot analysis was employed to determine the expressions of autophagy-associated markers and signaling pathway proteins. Autophagosomes were detected through mRFP-GFP-LC3 adenoviral transfection and transmission electron microscopy (TEM). Treatment of macrophages with the oxLDL/β2GPI/anti-β2GPI complex resulted in decreased expressions of Beclin1 and LC3 proteins, alongside an upregulation of SQSTM1/P62 protein expression. Additionally, there was a reduction in the number of autophagosomes and autolysosomes. An increase in the phosphorylation levels of phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) was also observed. Notably, the expressions of autophagy-associated markers were partially restored when the TLR4/NF-κB and PI3K/AKT/mTOR pathways were inhibited by their respective inhibitors. Our findings indicate that the oxLDL/β2GPI/anti-β2GPI complex inhibits macrophage autophagy in AS via the TLR4/NF-κB and PI3K/AKT/mTOR signaling pathways.

CKIP-1 inhibits M2 macrophage polarization to suppress the progression of gastric cancer by inactivating JAK/STAT3 signaling.

Gastric cancer (GC) is a frequently occurring malignancy with poor prognosis. Casein kinase 2 interacting protein-1 (CKIP-1) is a PH domain-containing protein implicated in regulating tumorigenesis and macrophage homeostasis. This study aimed to elucidate the role and potential mechanism of CKIP-1 in the progression of GC. CKIP-1 expression in GC tumor and para-carcinoma tissues was detected using RT-qPCR. Then, human monocyte cell line THP-1 was treated with PMA, interleukin (IL)-4 and IL-13 to induce M2-polarized macrophages. CD206, arginase-1 (Arg-1) and transforming growth factorβ1 (TGFβ1) expression in M2-polarized macrophages with or without CKIP-1 overexpression was evaluated. Moreover, GC cell lines (MKN45 and HGC27 cells) were co-cultured with CKIP-1-overexpressed M2-polarized macrophages, and the viability, migration and invasion of GC cells were measured. Additionally, immunoblotting assessed the expression of JAK/STAT3 signaling-related proteins and STAT3 agonist Colivelin was used to treat GC cells to perform the rescue experiments to analyze the changes of malignant phenotypes of GC cells. Results showed that CKIP-1 was downregulated in GC tissues and M2-polarized macrophages. CKIP-1 overexpression inhibited M2 macrophage polarization and decreased TGFβ1 secretion. Besides, elevated CKIP-1 expression in M2-polarized macrophages inhibited the viability, migration and invasion of GC cells. Furthermore, CKIP-1 overexpression inactivated JAK2/STAT3 signaling in GC cells by inhibiting TGFβ1 level. Specifically, Colivelin treatment abrogated the influences of CKIP-1 upregulation on the malignant phenotypes of GC cells. Collectively, CKIP-1 inhibits M2 macrophage polarization to suppress the progression of GC by inactivating JAK/STAT3 signaling pathway.

Treatment of polyethylene microplastics degraded by ultraviolet light irradiation causes lysosome-deregulated cell death

BackgroundMicroplastics (MPs), plastic particles < 5 mm in size, are prevalent in the environment, and human exposure to them is inevitable. To assess the potential risk of MPs on human health, it is essential to consider the physicochemical properties of environmental MPs, including polymer types, size, shape, and surface chemical modifications. Notably, environmental MPs undergo degradation due to external factors such as ultraviolet (UV) rays and waves, leading to changes in their surface characteristics. However, limited knowledge exists regarding the health effects of MPs, with a specific focus on their surface degradation. This study concentrates on cytotoxic MPs with surface degradation through UV irradiation, aiming to identify the mechanisms underlying their cell toxicity.ResultsPolyethylene (PE) and surface-degraded PE achieved through UV light irradiation were employed as model MPs in this study. We explored the impact of PE and degraded PE on cell death in murine macrophage cell line RAW264.7 cells and human monocyte cell line THP-1 cells. Flow cytometric analysis revealed that degraded PE induced programmed cell death without activating caspase 3, while non-degraded PE did not trigger programmed cell death. These findings suggest that degraded PE might induce programmed cell death through mechanisms other than caspase-driven apoptosis. To understand the mechanisms of cell death, we investigated how cells responded to degraded PE-induced cellular stress. Immunofluorescence and western blotting analyses demonstrated that degraded PE induced autophagosome formation and increased p62 expression, indicating inhibited autophagy flux after exposure to degraded PE. Furthermore, degraded PE exposure led to a decrease in acidic lysosomes, indicating lysosomal dysregulation. These results imply that degraded PE induces lysosomal dysfunction, subsequently causing autophagy dysregulation and cell death.ConclusionsThis study unveils that UV-induced degradation of PE results in cell death attributed to lysosomal dysfunction. The findings presented herein provide novel insights into the effects of surface-degraded MPs on biological responses.

Characterization of macrophages associated with human skin models exposed to UV radiation.

Skin macrophages play important roles in the response to external stimuli. Human skin equivalents (HSEs) incorporating the human monocytic cell line THP-1 were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and ultraviolet A (UVA) on macrophages. Transcriptomic analysis revealed that THP-1 cells in HSEs were enriched in extracellular matrix interaction hallmark but downregulated in DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs presented epidermal distortion and increased DNA double-strand breaks (DSBs). The genes associated with oxidative stress and the inflammatory response were significantly upregulated in THP-1 cells. When the photoprotective agent mycosporine-2-glycine from cyanobacteria was applied to HSEs, the incidence of UVA-induced DSBs was significantly lower, and inflammatory and UV response hallmarks were downregulated in THP-1 cells. Taken together, these results suggest that immunocompetent HSEs can be used to investigate the responses of skin-resident macrophages to external stimuli.

Increased CRHR1 expression on monocytes from patients with AA enables a pro-inflammatory response to corticotrophin-releasing hormone.

Stress may play a key role in alopecia areata (AA), though the exact interactions of stress with AA remain undefined. Corticotropin-releasing hormone (CRH), the proximal regulator of the stress axis, has been recognized as an immunomodulatory factor in tissues and peripheral blood mononuclear cells (PBMCs). We used multicolour flow cytometry to identify receptor CRHR1 expression on PBMC subsets in AA patients (n = 54) and controls (n = 66). We found that CRHR1 was primarily expressed by circulating monocytes. CRHR1 expression on monocytes was enhanced in AA compared with controls (3.17% vs. 1.44%, p = 0.002, chi-squared test). AA incidence was correlated to elevated CD14+ monocyte numbers (R = 0.092, p = 0.036) and markedly independently correlated with increased CRHR1 expression (R = 0.215, p = 0.027). High CRHR1 expression was significantly related to chronic AA (disease duration >1 year; p = 0.003, chi-squared test), and large lesion area (AA area >25%; p = 0.049, chi-squared test). We also observed enhanced percentages of active monocytes and reduced CD16+ CD3- NK cell numbers in AA patients' PBMCs (p = 0.010; 0.025, respectively). Invitro CRH treatment of PBMCs and human monocyte cell line THP-1 promoted CD86 upregulation. The findings imply that stress-related factors CRH and CRHR1 contribute to AA development and progression where higher CRHR1 expression is associated with chronic AA and larger lesions.

Cytokine secretion by in vitro cultures of lung epithelial cells, differentiated macrophages and differentiated dendritic cells incubated with pneumococci and pneumococcal extracellular vesicles.

Streptococcus pneumoniae is an important human pathogen that can colonize the respiratory tract of healthy individuals. The respiratory tract mucosa is thus the first barrier for this pathogen. In this study, we have tested three models of the respiratory epithelium with immune cells: (i) monolayer of A549 human lung epithelial cells, (ii) A549 + macrophages differentiated from the human monocytic THP-1 cell line (dMφ) and (iii) A549 + dMφ + dendritic cells differentiated from THP-1 (dDC) using a two-chamber system. Pneumococcal strains Rx1 (non-encapsulated) and BHN418 (serotype 6B) were incubated with the cells and secretion of IL-6, IL-8, IL-1β, TNF-α and IL-10 was evaluated. Overall, the models using co-cultures of A549 + dMφ and A549 + dMφ + dDC elicited higher levels of pro-inflammatory cytokines and the non-encapsulated strain elicited an earlier cytokine response. BHN418 pspA (pneumococcal surface protein A) and pspC (pneumococcal surface protein C) knockouts elicited similar cytokine secretion in the co-culture models, whereas BHN18 ply (pneumolysin) knockout induced much lower levels. The results are in accordance with the activation of the inflammasome by Ply. Finally, we evaluated pneumococcal extracellular vesicles (pEVs) in the co-culture models and observed secretion of pro-inflammatory cytokines in the absence of cytotoxicity. Since pEVs are being studied as vaccine candidate against pneumococcal infections, the co-cultures of A549 + dMφ and A549 + dMφ + dDC are simple models that could be used to evaluate pEV vaccine batches.

Abstract MP15: Macrophage PPARα is essential to increased immune function induced by angiotensin converting enzyme (ACE).

Mice with increased macrophage expression of angiotensin converting enzyme (ACE), called ACE 10/10 mice, have a profound increase of macrophage function in response to diverse immune challenges including tumors, infection, atherosclerosis, and Alzheimer’s disease. ACE 10/10 macrophages are highly efficient in oxidative ATP generation, and they express increased amounts of peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor that controls lipid metabolism. To pinpoint the role of PPARα in increasing macrophage immune function, we used LysM-Cre-LoxP system to generate ACE 10/10 mice selectively lacking macrophage PPARα (A10-PPARα-Cre mice). This reduced macrophage fatty acid metabolism, reduced oxidative metabolism, and lowered macrophage intracellular ATP. Functionally, A10-PPARα-Cre macrophage are impaired in both adaptive and innate immunity. A10-PPARα-Cre mice have larger tumor growth of B16-F10 melanoma, less intratumor macrophages, reduced anti-tumor cytokines, and reduced generation of tumor antigen-specific CD8 + T cells. The mice also have a very attenuated anti-bacterial response to methicillin-resistant Staphylococcus aureus (MRSA) infection, with more severe MRSA tissue colonization in vivo . In addition, increasing ACE expression or adding a PPARα agonist to the human monocytic cell line THP-1 enhances tumor cell cytotoxicity, phagocytosis of bacteria, and bacterial killing. Thus, we find that elevated macrophage PPARα is absolutely required for the increased immunity of ACE 10/10 mice. This may indicate a pathway to increasing human immunity.

Inhibitory Effect of Pyra-Metho-Carnil on the Differentiation and Maturation of Macrophages in Normal and Cancer Microenvironments.

In a previous study, we have demonstrated heightened Pyra-Metho-Carnil (PMC) efficacy in nude mice with intact innate immunity that lack T and B cells. This has prompted hypothesizing that PMC may target macrophages that promote cancer growth. In this study, we conducted co-culture experiments with macrophages derived from THP-1 human monocyte cell lines and spheroids representing normal and cancer microenvironments. We then performed RNA sequencing and flow cytometry analysis to elucidate the mechanisms by which PMC affects macrophage differentiation and maturation. THP-1 cells were differentiated by phorbol 12-myristate 13-acetate (PMA) and matured by PMA and lipopolysaccharide (LPS) either with or without PMC. Co-cultures were performed using stimulated THP-1 cells and HKe3-wild-type KRAS or HKe3-mutant (mt) KRAS spheroids. We then performed RNA-seq analysis of THP-1 cells stimulated by PMA (either with or without PMC) and flow cytometry analysis of mice peripheral blood obtained after PMC administration. THP-1 cells matured by PMA and LPS specifically increased the area of HKe3-mtKRAS cancer spheroids and the addition of PMC to THP-1 cells was found to inhibit cancer spheroid growth. RNA-seq data suggested that PMC treatment of THP-1 cells stimulated with PMA suppressed cell motility regulatory functions via down-regulation of the NF[Formula: see text]B pathway. Furthermore, flow cytometry results showed that PMC treatment suppressed monocyte maturation in B6 mice. The high level of in vivo tumor suppression caused by PMC may be due to inhibition of the differentiation and maturation of tumor-associated macrophages via the NF[Formula: see text]B signaling pathway.

Carbon Nanotube Immunotoxicity in Alveolar Epithelial Type II Cells Is Mediated by Physical Contact-Independent Cell-Cell Interaction with Macrophages as Demonstrated in an Optimized Air-Liquid Interface (ALI) Coculture Model.

There is a need for the assessment of respiratory hazard potential and mode of action of carbon nanotubes (CNTs) before their approval for technological or medical applications. In CNT-exposed lungs, both alveolar macrophages (MФs), which phagocytose CNTs, and alveolar epithelial type II cells (AECII cells), which show tissue injury, are impacted but cell-cell interactions between them and the impacted mechanisms are unclear. To investigate this, we first optimized an air-liquid interface (ALI) transwell coculture of human AECII cell line A549 (upper chamber) and human monocyte cell line THP-1 derived macrophages (lower chamber) in a 12-well culture by exposing macrophages to CNTs at varying doses (5-60 ng/well) for 12-48 h and measuring the epithelial response markers for cell differentiation/maturation (proSP-C), proliferation (Ki-67), and inflammation (IL-1β). In optimal ALI epithelial-macrophage coculture (3:1 ratio), expression of Ki-67 in AECII cells showed dose dependence, peaking at 15 ng/well CNT dose; the Ki-67 and IL-1β responses were detectable within 12 h, peaking at 24-36 h in a time-course. Using the optimized ALI transwell coculture set up with and without macrophages, we demonstrated that direct interaction between CNTs and MФs, but not a physical cell-cell contact between MФ and AECII cells, was essential for inducing immunotoxicity (proliferative and inflammatory responses) in the AECII cells.

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Macropinocytosis Is the Principal Uptake Mechanism of Antigen-Presenting Cells for Allergen-Specific Virus-like Nanoparticles.

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

1,25-dihydroxyvitamin D3 augments low-dose PMA-based monocyte-to-macrophage differentiation in THP-1 cells

The human monocytic THP-1 cell line is the most routinely employed in vitro model for studying monocyte-to-macrophage differentiation. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (1,25D3) vary drastically between studies. Given that differences in differentiation protocols have the potential to impact the characteristics of the macrophages produced, we aimed to assess the efficacy of three different THP-1 differentiation protocols by assessing changes in morphology and gene- and cell surface macrophage marker expression. THP-1 cells were differentiated with either 5 nM PMA, 10 nM 1,25D3, or a combination thereof, followed by a rest period. The results indicated that all three protocols significantly increased the expression of the macrophage markers, CD11b (p < 0.001) and CD14 (p < 0.010). Despite this, THP-1 cells exposed to 1,25D3 alone did not adopt the morphological and expression characteristics associated with macrophages. PMA was required to produce these characteristics, which were found to be more pronounced in the presence of 1,25D3. Both PMA- and PMA with 1,25D3−differentiated THP-1 cells were capable of M1 and M2 macrophage polarization, though the gene expression of polarization-associated markers was most pronounced in PMA with 1,25D3−differentiated THP-1 cells. Moreover, the combination of PMA with 1,25D3 appeared to support the process of commitment to a particular polarization state.

Non-invasive Physical Plasma Modulates Macrophage Polarization: A Potential Strategy for Tumor Microenvironment Remodeling.

Non-invasive physical plasma (NIPP) has shown promise in the treatment of cancer. However, conflicting results have been reported regarding the effect of NIPP on macrophage polarization. As tumor-associated macrophages (TAMs) are essential in the regulation of cancer development, this study aimed to determine the role of NIPP treatment in macrophage polarization and tumor-microenvironment (TME) remodeling. A portable NIPP device, Plasma Care (Terraplasma Medical, Garching, Germany), was employed as the source of NIPP. The human monocytic cell line THP-1 was adopted as the cell model for macrophage differentiation and polarization. The effects of NIPP treatment on temperature, pH value, and oxidative stress induction of the culture medium were examined to validate the feasibility of applying the NIPP device in subsequent cell treatment. The changes in morphology, viability, and proliferation of THP-1 cells after NIPP treatment were determined. The expression of M1/M2 macrophage markers was examined by real-time quantitative polymerase chain reaction. No significant changes were observed in temperature and pH value after NIPP treatment, while the formation of hydrogen peroxide was promoted in a time-dependent manner. Cell morphology, viability, and proliferation were not affected by up to 6 minutes of NIPP treatment. In monocytes, 6 minutes of NIPP treatment significantly increased the expression of M1 markers (TNF-α and IL-6) and suppressed the M2 marker (CD206), findings which were consistent in the monocyte-derived macrophages. Furthermore, NIPP treatment also significantly promoted M1 polarization in the monocyte-derived macrophages induced by phorbol 12-myristate 13-acetate. NIPP is a safe and robust oxidative stress inducer and showed potential in TAM regulation by promoting M1 macrophage polarization.

Decreased atherosclerosis and abdominal aortic aneurysm in mice deficient in polymeric immunoglobulin receptor

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): La Caixa Banking Foundation and Comunidad de Madrid Background Polymeric immunoglobulin receptor (PIGR) is a transmembrane protein involved in the transcytosis of polymeric immunoglobulins that is widely expressed in mucosal epithelial cells. Recent data from our group described increased PIGR levels in plasma and tissue of atherosclerotic subjects. However, the role for PIGR in the mechanisms involved in vascular remodeling has not been addressed. Purpose To analyse the role for PIGR in experimental mouse models of atherosclerosis and abdominal aortic aneurysm (AAA). Methods We analyzed PIGR expression, secretion and distribution in early atherosclerotic plaques and AAA tissues compared to healthy aortic samples, as well as in macrophages derived from the human monocytic THP-1 cell line. Next, we studied the effect of global PIGR deficiency in experimental atherosclerosis (Ldlr-/- Pigr-/- mice fed with atherogenic diet for 10 weeks). In addition, the contribution of hematopoietic PIGR was assessed in both experimental atherosclerosis and AAA (Ldlr-/- mice fed with atherogenic diet and 1 µg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments. Finally, bone marrow-derived macrophages (BMDMs) from Pigr-/- and control mice were isolated to assess their inflammatory expression profile and modulation of polarization in response to oxidized LDL (ox-LDL), as well as their ability to promote foam cell formation. Results PIGR expression was increased in the intima of early human atherosclerotic lesions and showed colocalization with CD68+ expression. PIGR expression was also increased during the in vitro differentiation of THP-1 monocytes to macrophages with phorbol myristate acetate. Both global and hematopoietic-specific PIGR deficient mice displayed reduced plaque size in the aortic sinuses as well as a strong decrease in macrophage infiltration (CD68+ cells), while no changes in smooth muscle cells (α-SMA+ cells) and collagen content were observed. Regarding AAA, increased PIGR levels were found in human AAA tissues, associated with infiltrating CD68+ cells. PIGR deficiency in hematopoietic cells also showed significantly reduced AAA dilatation and reduced macrophage infiltration. In vitro experiments with BMDMs obtained from Pigr-/- mice and stimulated with ox-LDL showed reduced proinflammatory gene expression (TNF-a and IL-6) and M1 markers (iNOS and CD11c) as well as diminished foam cell formation. Conclusions PIGR deficiency in hematopoietic cells decreases atherosclerosis and AAA progression by modulating macrophage inflammatory responses, suggesting a novel proinflammatory role for PIGR in vascular pathologies. Future experiments will test the potential therapeutic effect of modulating PIGR to dampen the inflammatory profile in vascular diseases.Results in human and mouse studies

Critical view on antimicrobial, antibiofilm and cytotoxic activities of quinazolin-4(3H)-one derived schiff bases and their Cu(II) complexes

A series of nine 2,3-disubstituted-quinazolin-4(3H)-one derived Schiff bases and their three Cu(II) complexes was prepared and tested for their antimicrobial activities against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the substances were tested in vitro against Mycobacterium tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 and M. smegmatis ATCC 700084. While anti-enterococcal and antimycobacterial activities were insignificant, 3-[(E)-(2-hydroxy-5-nitrobenzylidene)amino]-2-(2-hydroxy-5-nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (SB3) and its Cu(II) complex (SB3–Cu) demonstrated bacteriostatic antistaphylococcal activity. In addition, both compounds, as well as the other two prepared complexes, showed antibiofilm activity, which resulted in a reduction of biofilm formation and eradication of mature S. aureus biofilm by 80% even at concentrations lower than the values of their minimum inhibitory concentrations. In addition, the compounds were tested for their cytotoxic effect on the human monocytic leukemia cell line THP-1. The antileukemic efficiency was improved by the preparation of Cu(II) complexes from the corresponding non-chelated Schiff base ligands.

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

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. 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. 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. 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.

Ethnobotanical survey and biological activities of plants used for cancer treatment in traditional Senegalese medicine

Female breast cancer is known to be one of the leading causes of death in Senegal. In Senegal, the absence of a national cancer control program, the lack of specialized infrastructure and qualified human resources and the exorbitant cost of care have contributed to the extensive use of traditional medecine, particularly in rural areas. This study aims to inventory the medicinal plants used by these healers and to assess the cytotoxic and antioxidant activities of the most widely used one. Data on healers and their use practices and information on plants were collected through the administration of a structured questionnaire. Based on their citation frequencies during the survey, Antiaris Africana Engler, Hymenocardia Acida Tul. and Halouf Halal (local name) were selected for chemical and biological studies. Their hydroalcoholic extracts were analyzed in terms of antioxidant capacity and cytotoxic effects, again, in the human cancer cell line. The study revealed a total of 65 medicinal plants belonging to 35 different families. The plant parts used by traditional healers are leaves (63.89%), roots (11.11%), bark (15.28%), fruits (2.78%), and others (6.94%). Generally, herbal medicine is prepared as a powder and mixed with water by maceration (55.38%) and administered orally. A. Africana ranked first with a citation frequency of 5.7% and its hydroalcoholic extract had the highest antioxidant activity in TEAC (6533.64±7 μmol ET/g dry plant) and in ORAC (3745.17±4.8 μmol ET/g dry plant) followed by H. Acida in TEAC (3115.6±145 μmol ET/g dry plant) and in ORAC (4105.29±872 μmol ET/g dry plant). The hydroalcoholic extract of A. Africana exhibited the highest cytotoxic activity in MCF-7 (Human mammary) and THP-1 (Human acute monocytic leukemia cell line) but had low activity against HTC-116 (Human carcinoma colorectal) and A-375 (Human skin malignant melanoma). The percentages of proapoptotic cells were, respectively, 68.85±6.22, 58.1±1.90 and 48.58±1.4%. These results provide scientific support for the traditional use of medicinal plants in cancer treatment and constitute a database for biological screening to isolate cytotoxic plant-based molecules.

Colletodiol derivatives of the endophytic fungus Trichocladium sp.

The OSMAC (one strain many compounds) concept is a cultivation-based approach to increase the diversity of secondary metabolites in microorganisms. In this study, we applied the OSMAC-approach to the endophytic fungus Trichocladium sp. by supplementation of the cultivation medium with 2.5% phenylalanine. This experiment yielded five new compounds, trichocladiol (1), trichocladic acid (2), colletodiolic acid (3), colletolactone (4) and colletolic acid (5), together with five previously described ones (6–10). The structures were elucidated via comprehensive spectroscopic measurements, and the absolute configurations of compound 1 was elucidated by using TDDFT-ECD calculations. For formation of compounds 3–5, a pathway based on colletodiol biosynthesis is proposed. Compound 6 exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.78 μM as well as a strong cytotoxic effect against the human monocytic cell line THP1 with an IC50 of 0.7 μM. Compound 8 showed moderate antibacterial activity against Mycobacterium tuberculosis with a MIC of 25 μM and a weak cytotoxic effect against THP1 cells with an IC50 of 42 μM.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions. Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

1,8-Cineol Attenuates Checkpoint Molecule PDL-1 and Adhesion Molecule CX3CR1 in Circulating Monocytes in Otitis Media Patients.

Peripheral blood monocytes can be subdivided into different subsets based on the CD14/CD16 surface characteristics. Monocytes are a major source of cytokine secretion of pro-inflammatory immune responses, whereas CD16+ monocyte subsets can also contribute to persistent inflammation in the context of chronic diseases. However, the regulation and cellular characteristics of circulating monocyte subsets in patients with chronic otitis media (COM), one of the largest public health burdens, remains largely unknown. In this study, we analyzed individual distributions of circulating monocyte subsets and associated protein expression levels of adhesion protein and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), and CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor), as well as checkpoint molecule PD-L1 (programmed cell death ligand-1), in a gender-balanced cohort of 14 patients with chronic otitis media using flow cytometry, especially in view of the therapeutic impact of the natural plant-derived monoterpene oxide 1,8-Cineol. Furthermore, using the human monocyte cell line THP-1 as a model, we investigated the influence of anti-inflammatory 1,8-Cineol on monocytic cytokine secretion patterns using human cytokine arrays and ELISA measurements. The data revealed significantly elevated expression levels of all analyzed adhesion molecules in certain monocyte subsets in COM patients; CX3CR1 was especially significantly down-regulated in response to 1,8-Cineol administration. Moreover, the data revealed significantly increased monocytic PD-L1 expression levels in circulating classical and intermediate monocyte subsets from COM patients compared to healthy donors, but also a significant decrease in PD-L1 in intermediate monocytes upon 1,8-Cineol therapy compared to the pre-treatment situation. Furthermore, the increased secretion of cytokine CXCL10 by THP-1 monocytes in response to LPS was found to be strongly attenuated by 1,8-Cineol. Plasma levels of CXCL10 were also significantly increased in COM patients, but no significant differences between the pre and post 1,8-Cineol situation were observed. The present study revealed new insights into the bioactive anti-inflammatory effects of 1,8-Cineol in terms of monocyte adhesion and immune regulation. Our data suggest the potential role of cytokine CXCL10 in COM development and maintenance, which is also involved in the activity of its concomitant disease, rheumatoid arthritis.