Human Monocytic Cell Research Articles

Investigating DAGLβ expression and activity in human monocytes via proteomic and lipidomic profiling

Abstract Lipid-metabolizing enzymes are important targets for small-molecule inhibitor discovery as the regulation of inflammation responses have been linked to lipid metabolism cascades controlled by these enzymes. Non-steroidal anti-inflammatory drugs inhibit enzymes in the COX pathway preventing conversion of arachidonic acid (AA) into inflammation-inducing prostaglandins. An upstream precursor to AA is the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which is biosynthesized from the cleavage of arachidonate-containing diacylglycerols (DAGs) by diacylglycerol lipase beta (DAGLβ). Previous work to identify DAGLβ’s role in inflammatory function led to the creation of KT109, a DAGLβ-specific 1,2,3-triazole urea inhibitor. Primary mouse macrophages treated with KT109 showed reductions in AA and 2-AG and an increase in arachidonate-containing DAGs, demonstrating that DAGLβ is key in proinflammatory lipid regulation and signaling. These studies focused on the effects of inhibiting mouse DAGLβ, but information regarding KT109 inhibition of human DAGLβ is still lacking. Herein, we applied activity-based protein profiling (ABPP) and quantitative proteomic and lipidomic methods to determine the effectivity of KT109 inhibition in human monocytic cell line THP1. Potency (IC 50values) for DAGLβ inhibition by KT109 in human THP1 cells were comparable to the magnitude of inhibition in mouse cells. Lipidomic analysis of KT109-treated monocytic and macrophage-differentiated THP1s did not demonstrate an accumulation of arachidonate-containing DAGs. These findings show a potential difference in the expression and activity of human DAGLβ when compared to mouse DAGLβ and additional targets of KT109 in human cells. Funding and disclosures: This work was supported by the National Institute on Drug Abuse grant no. R01 DA043571.

Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway

BackgroundClonorchis sinensis granulin (CsGRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of CsGRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of CsGRN on the malignant transformation of HIBECs and its possible underlying mechanism.MethodsThe malignant transformation phenotypes of HIBECs after CsGRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of CsGRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in CsGRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in CsGRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs.ResultsExcessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with CsGRN. The expression of the markers of M2 macrophages significantly increased in CsGRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with CsGRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the CsGRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in CsGRN-treated HIBECs and further repressed the malignant transformation of HIBECs.ConclusionsOur results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, CsGRN promoted the malignant transformation of the latter.Graphical abstract

SVEP1 influences monocyte to macrophage differentiation via integrin α4β1/α9β1 and Rho/Rac signalling

BackgroundThe large extracellular matrix protein SVEP1 mediates cell adhesion via integrin α9β1. Recent studies have identified an association between a missense variant in SVEP1 and increased risk of coronary artery disease (CAD) in humans and in mice Svep1 deficiency alters the development of atherosclerotic plaques. However how SVEP1 functionally contributes to CAD pathogenesis is not fully understood. Monocyte recruitment and differentiation to macrophages is a key step in the development of atherosclerosis. Here, we investigated the requirement for SVEP1 in this process. MethodsSVEP1 expression was measured during monocyte–macrophage differentiation in primary monocytes and THP-1 human monocytic cells. SVEP1 knockout THP-1 cell lines and the dual integrin α4β1/α9β1 inhibitor, BOP, were utilised to investigate the effect of these proteins in THP-1 cell adhesion, migration and cell spreading assays. Subsequent activation of downstream integrin signalling intermediaries was quantified by western blotting. ResultsSVEP1 gene expression increases in monocyte to macrophage differentiation in human primary monocytes and THP-1 cells. Using two SVEP1 knockout THP-1 cells we observed reduction in monocyte adhesion, migration, and cell spreading compared to control cells. Similar results were found with integrin α4β1/α9β1 inhibition. We demonstrate reduced activity of Rho and Rac1 in SVEP1 knockout THP-1 cells. ConclusionsSVEP1 regulates monocyte recruitment and differentiation phenotypes through an integrin α4β1/α9β1 dependent mechanism. General significanceThese results describe a novel role for SVEP1 in monocyte behaviour relevant to CAD pathophysiology.

Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest.

Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-β-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.

932 Integrin α5 regulates dynamics and motility of human monocyte-derived langerhans cells during immune response

932 Integrin α5 regulates dynamics and motility of human monocyte-derived langerhans cells during immune response

Nitrobenzoates and Nitrothiobenzoates with Activity against M. tuberculosis.

Esters of weak acids have shown improved antimycobacterial activity over the corresponding free acids and nitro benzoates in particular have previously shown to have a very intriguing activity. To expand the potential of nitro-derivatives of benzoic acid as antimycobacterial drugs and explore the effects of various structural features on the activity of these compounds, we have obtained a library of 64 derivatives containing esters and thioesters of benzoates and studied their activity against M. tuberculosis, the stability of the compounds, their activation by mycobacterial enzymes and the potential cytotoxicity against human monocytic THP-1 cell line. Our results showed that the most active compounds are those with an aromatic nitro substitution, with the 3,5-dinitro esters series being the most active. Also, the greater antitubercular activity for the nitro derivatives was shown to be unrelated to their pKa values or hydrolysis rates. Given the conventional relationship between nitro-containing substances and toxicity, one might anticipate that the great antimicrobial activity of nitro compounds would be associated with high toxicity; yet, we have not found such a relationship. The nitrobenzoate scaffold, particularly the 3,5-dinitrobenzoate scaffold, merits further investigation, because it has the potential to generate future antimycobacterial agents with improved activity.

Activation of the High Mobility Group Box 1/Receptor for Advanced Glycation Endproducts /NOD-like Receptor Family Pyrin Domain-Containing 3 Axis Under Chronic Intermittent Hypoxia Induction Promotes the Progression of Atherosclerosis in ApoE-/- Mice.

Background Chronic intermittent hypoxia (CIH) has been regarded as an important cause of atherosclerotic disease. In our study, we set out to investigate whether CIH regulated the high mobility group box 1/receptor for advanced glycation endproducts/NOD-like receptor family pyrin domain-containing 3 (HMGB1/RAGE/NLRP3) axis to affect the progression of atherosclerosis. Methods and Results Initially, peripheral blood samples were collected from patients with single obstructive sleep apnea, atherosclerosis complicated with obstructive sleep apnea, and healthy volunteers. In vitro cell experiments were conducted using human monocyte cell line THP-1 and human umbilical vein endothelial cells to explore the role of HMGB1 in cell migration, apoptosis, adhesion, and transendothelial migration. In addition, a CIH-induced atherosclerosis mouse model was established for further identifying the critical role of the HMGB1/RAGE/NLRP3 axis in atherosclerosis. Upregulated HMGB1 and RAGE were found in patients with atherosclerosis complicated with obstructive sleep apnea. CIH induction increased HMGB1 expression by inhibiting HMGB1 methylation, activating the RAGE/NLRP3 axis. After inhibition of the HMGB1/RAGE/NLRP3 axis, monocyte chemotaxis and adhesion were repressed, and macrophage-derived foam cell formation was inhibited, accompanied by suppression of endothelial and foam cell apoptosis and inflammatory factor secretion. In vivo animal experiments also noted that the progression of atherosclerosis was prevented by inhibition of the HMGB1/RAGE/NLRP3 axis in CIH-induced ApoE-/- mice. Conclusions Taken together, CIH induction can upregulate HMGB1 through inhibition of HMGB1 methylation, which activates the RAGE/NLRP3 axis to promote inflammatory factor secretion, thereby promoting the progression of atherosclerosis.

Comprehensive proteomic analysis of autophagosomes derived from Leishmania-infected macrophages.

Recently, autophagy has been implicated as a host defense mechanism against intracellular pathogens. On the other hand, certain intracellular pathogens such as Leishmania can manipulate the host's autophagy to promote their survival. Our recent findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of regulation by the mammalian target of rapamycin complex 1. This suggests the fine-tuning of autophagy to optimally promote parasite survival, possibly by the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Selected proteomic results were validated by Western blotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes, while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level. A comprehensive analysis of the Leishmania-induced autophagosome proteome will be instrumental in the advancement of understanding leishmaniasis.

Modification of polypropylene mesh by titanium compound: An in Vivo and in Vitro study

Objective: Previous basic studies on the use of titanized polypropylene meshes in abdominal external hernia repair are not only limited, but also highly controversial. This study aims to investigate the modification effect of titanium compounds on polypropylene materials and compare the performance of two kinds of meshes both in vivo and in vitro.Methods: Human peritoneal mesothelial cells (HMrSV5), human epidermal fibroblasts (HSF), and human monocytic cells (THP-1) were cultured in vitro to simulate the abdominal external hernia environment. Titanized polypropylene meshes (Ti) and polypropylene mesh (Non-Ti) were co-cultured with the cells respectively. The effects of titanium compounds on cell growth were determined by cell activity and apoptosis, and the growth of cells on the mesh surface was assessed using a scanning electron microscope and a confocal microscope. In vivo experiments, different sizes titanized polypropylene meshes and polypropylene meshes were placed between the external oblique abdominal muscle and the internal oblique abdominal muscle, the parietal peritoneum, the serous layer of the descending colon, and the underside of the femoral nerve in rabbits. The effects of titanium compounds were evaluated by observing the anti-adhesion, anti-contraction, anti-fibrotic properties, and effects on nerves of the mesh.Results: Titanium compounds effectively reduced the effects of polypropylene material on cell growth, and improved the fixation and adhesion of HMrSV5, HSF and THP-1 (M0) on the surface of titanized polypropylene meshes. Furthermore, titanium compounds improved the adhesion, contraction and fibrosis of polypropylene material, as well as reduced nerve damage. This improvement demonstrated a regular trend with the type of titanized polypropylene meshes.Conclusion: The titanium compounds improved the biocompatibility of the polypropylene material, which was conducive to the fixation and adhesion of cells on the surface of the meshes, and alleviated the adhesion and contraction of the meshes, and the degree of tissue fibrosis, as well as the influence on nerves.

Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis.

Upon migrating into the tissues, hematopoietic stem cell (HSC)-derived monocytes differentiate into macrophages, playing a crucial role in determining innate immune responses towards external pathogens and internal stimuli. However, the regulatory mechanisms underlying monocyte-to-macrophage differentiation remain largely unexplored. Here we divulge a previously uncharacterized but essential role for an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), in monocyte-to-macrophage maturation. FLRT2 is almost undetectable in human monocytic cell lines, human peripheral blood mononuclear cells (PBMCs), and mouse primary monocytes but significantly increases in fully differentiated macrophages. Myeloid-specific deletion of FLRT2 (Flrt2ΔMyel ) contributes to decreased peritoneal monocyte-to-macrophage generation in mice in vivo, accompanied by impaired macrophage functions. Gain- and loss-of-function studies support the promoting effect of FLRT2 on THP-1 cell and human PBMC differentiation into macrophages. Mechanistically, FLRT2 directly interacts with Unc-5 netrin receptor B (UNC5B) via its extracellular domain (ECD) and activates Akt/mTOR signaling. In vivo administration of mTOR agonist MYH1485 reverses the impaired phenotypes observed in Flrt2ΔMyel mice. Together, these results identify FLRT2 as a novel pivotal endogenous regulator of monocyte differentiation into macrophages. Targeting the FLRT2/UNC5B-Akt/mTOR axis may provide potential therapeutic strategies directly relevant to human diseases associated with aberrant monocyte/macrophage differentiation.

Abstract 4070: Engineering TRAIL-expressing immune cells to target GI malignancies

Abstract While adoptive cell therapies, such as chimeric antigen receptor (CAR) T cells, have had promising results in hematologic malignancies, their translation to solid tumors has been mostly unsuccessful. A significant limitation to use of adoptive cell therapy in solid tumors has been life-threatening off-target toxicities, including acute respiratory toxicity and cytokine release syndrome. The native T cell program releases a diverse range of toxic proteins upon activation, including interferons, interleukins, perforins, and granzymes. Rather than activate the native T-cell program, selective expression of tumor-targeting agents may be advantageous. TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively target tumor cells with minimal toxicity to normal tissues. We therefore engineered Jurkat (a human CD4+ T cell leukemia cell line) and THP-1 (a human monocytic leukemia cell line) cells to constitutively express wild-type TRAIL using a third-generation lentivirus system. These cells killed multiple cell lines across colorectal and pancreatic cancer, thus representing a possible therapeutic strategy to target GI malignancies. Furthermore, the addition of the ferroptosis induced erastin enhanced cytotoxicity of TRAIL-engineered cells, suggesting that targeting ferroptosis in addition to apoptosis is a promising strategy for enhancing immune killing. Future work will focus on overcoming TRAIL resistance, tumor trafficking, and alteration of the tumor microenvironment. Citation Format: Praveen R. Srinivasan, Shengliang Zhang, Wafik S. El-Deiry. Engineering TRAIL-expressing immune cells to target GI malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4070.

Development of Curcumin and Piperine-Loaded Bio-Active Self-Nanoemulsifying Drugs and Investigation of Their Bioactivity in Zebrafish Embryos and Human Hematological Cancer Cell Lines.

Curcumin (CUR) and piperine (PP) are bioactive compounds with prominent pharmacological activities that have been investigated for the treatment of various diseases. The aim of the present study is to develop Bio-SNEDDS for CUR and PP as a combined delivery system for cancer therapy. CUR and PP loaded Bio-SNEDDSs with varying compositions of bioactive lipid oils, surfactants, and cosolvents were prepared at room temperature. Bio-SNEDDSs were characterized using a Zetasizer Nano particle size analyzer and further examined by transmission electron microscopy (TEM) for morphology. The in vivo toxicity of the preparations of Bio-SNEDDS was investigated in wild-type zebrafish embryos and cytotoxicity in THP-1 (human leukemia monocytic cells), Jurkat (human T lymphocyte cells) and HUVEC (non-cancerous normal) cells. Bio-SNEDDSs were successfully developed with black seed oil, Imwitor 988, Transcutol P and Cremophor RH40 at a ratio of 20/20/10/50 (%w/w). The droplet size, polydispersity index and zeta potential of the optimized Bio-SNEDDS were found to be 42.13 nm, 0.59, and -19.30 mV, respectively. Bio-SNEDDS showed a spherical structure evident by TEM analysis. The results showed that Bio-SNEDDS did not induce toxicity in zebrafish embryos at concentrations between 0.40 and 30.00 μg/mL. In TG (fli1: EGFP) embryos treated with Bio-SNEDDS, there was no change in the blood vessel structure. The O-dianisidine staining of Bio-SNEDDS treated embryos at 48 h post-fertilization also showed a significant reduction in the number of blood cells compared to mock (DMSO 0.1% V/V) treated embryos. Bio-SNEDDS induced significant levels of cytotoxicity in the hematological cell lines THP-1 and Jurkat, while low toxicity in normal HUVEC cell lines was observed with IC50 values of 18.63±0.23 μg/mL, 26.03 ± 1.5 μg/mL and 17.52 ± 0.22 μg/mL, respectively. Bio-SNEDDS exhibited enhanced anticancer activity and could thus be an important new pharmaceutical formulation to treat leukemia.

HMG-CoA Reductase Inhibitors Suppress Monosodium Urate-Induced NLRP3 Inflammasome Activation through Peroxisome Proliferator-Activated Receptor-γ Activation in THP-1 Cells.

Peroxisome proliferator-activated receptor γ (PPAR-γ) is thought to negatively regulate NLRP3 inflammasome activation. The aim of this study was to identify the inhibitory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on monosodium urate (MSU) crystal-induced NLRP3 inflammasome activation through the regulation of PPAR-γ in THP-1 cells. The expression of PPAR-γ, NLRP3, caspase-1, and interleukin-1β (IL-1β) in human monocytic THP-1 cells transfected with PPAR-γ siRNA or not and stimulated with MSU crystals was assessed using quantitative a real time-polymerase chain reaction and Western blotting. The expression of those markers in THP-1 cells pretreated with statins (atorvastatin, simvastatin, and mevastatin) was also evaluated. Intracellular reactive oxygen species (ROS) were measured using H2DCF-DA and flow cytometry analyses. THP-1 cells treated with MSU crystals (0.3 mg/mL) inhibited PARR-γ and increased NLRP3, caspase-1, and IL-1β mRNA and protein expression, and all those changes were significantly reversed by treatment with atorvastatin, simvastatin, or mevastatin. PPAR-γ activity revealed that MSU crystals suppressed PPAR-γ activity, which was markedly augmented by atorvastatin, simvastatin, and mevastatin. Transfecting cells with PPAR-γ siRNA attenuated the inhibitory effect of statins on MSU crystal-mediated NLRP3 inflammasome activation. Statins also significantly reduced the intracellular ROS generation caused by stimulation with MSU crystals. The inhibitory effects of atorvastatin and simvastatin on intracellular ROS generation were reduced in THP-1 cells transfected with PPAR-γ siRNA. This study demonstrates that PPAR-γ is responsible for suppressing MSU-mediated NLRP3 inflammasome activation. The inhibitory effect of statins on MSU-induced NLRP3 inflammasome activation depends on PPAR-γ activity and production and the inhibition of ROS generation.

Adjuvant action of needle-shaped BC microfibrils.

Bacterial cellulose (BC) is an unbranched biopolymer produced by microorganisms and composed of glucopyranose units linked by β-1,4 bonds. This study investigates the adjuvant action of needle-shaped BC microfibrils (BCmFs) in vitro using bovine serum albumin (BSA) as a model antigen. BC produced by the static culture of Komagataibacter xylinus was then microparticled (1-5μm) by acid hydrolysis and characterized using Dynamic Light Scattering and Scanning Electron Microscopy. Subsequently, Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy, cytotoxicity, TNF-α (tumour necrosis factor-alpha) and IL-6 (interleukin-6) cytokine secretion, and cellular uptake of the BCmFs-BSA conjugate on the human monocyte cell line (U937) differentiated into macrophages were performed. The microfibrils were determined to be 1-5μm in size, needle-shaped, with a zeta potential of - 32mV. Their conjugation with the model antigen, BSA, was demonstrated by FTIR analysis. In the cytotoxicity assay, BCmFs-BSA in macrophage cells showed high viability (over 70%). Although the highest TNF-α cytokine level (113pg/ml) was obtained with BCmFs-BSA (Bovine serum albumin) conjugate (500µg/ml) and was statistically significant (p = 0.0001) compared to the positive control group (BSA-aluminium hydroxide), IL-6 cytokine levels were not statistically different from those in the control group as desired. It has been shown in macrophage-differentiated U937 cells that microbially synthesized BC in the form of needle-shaped microfibrils (BCmFs) has a high cellular uptake capacity and increases the immunogenicity of the antigen. These results demonstrate for the first time that BCmFs have the potential to serve as a vaccine adjuvant.

Tumor-associated macrophages facilitate oral squamous cell carcinomas migration and invasion by MIF/NLRP3/IL-1β circuit: A crosstalk interrupted by melatonin

Invasion and migration are significant challenges for treatment of oral squamous cell carcinomas (OSCCs). Tumor-associated macrophages (TAMs) interact with cancer cells and are involved in tumor progression. Our recent study demonstrated that melatonin inhibits OSCC invasion and migration; however, the mechanism by which melatonin influences crosstalk between TAMs and OSCCs is poorly understood. In this study, a co-culture system was established to explore the interactions between human monocytic cells (THP-1 cells) and human tongue squamous cell carcinoma cells (SCC-15 cells). The results were verified using monocyte-derived macrophages (MDMs) isolated and differentiated from primary peripheral blood mononuclear cells. In vivo, assays were performed to confirm the anticancer effects of melatonin. SCC-15 cells co-cultured with THP-1 cells or MDMs exhibited increased migration and invasion, which was reversed by melatonin. Co-culture also increased the expression of macrophage migration inhibitory factor (MIF), CD40, CD163 and IL-1β, and these changes were also reversed by melatonin. Moreover, IL-1β secretion in THP-1 cells was MIF- and NLR family pyrin domain-containing 3 (NLRP3)-dependent, and treated with IL-1β enhanced the invasion and migration of SCC-15 cells. Furthermore, melatonin treatment significantly decreased tumor volumes and weights, and tumors from mice treated with melatonin had lower levels of MIF, NLRP3, and IL-1β than tumor from control mice. These results demonstrate that macrophages facilitate the progression of OSCCs by promoting the MIF/NLRP3/IL-1β signaling axis, which can be interrupted by melatonin. Therefore, melatonin could act as an alternative anticancer agent for OSCCs by targeting this signaling axis.

Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line

BackgroundAutophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy.MethodsIn this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed.ResultsThe results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6.ConclusionTaken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.

The NLRP3 Inflammasome Works as a Sensor for Detecting Hypoactivity of the Mitochondrial Src Family Kinases.

Gefitinib (GF), the tyrosine kinase inhibitor (TKI) targeting epidermal growth factor receptor, initiates lung inflammation through the NLR family pyrin domain containing 3 (NLRP3) inflammasome. However, the molecular targets and mechanisms underlying the inflammatory action of GF remain unknown. In this study, we identified mitochondrial Src family kinases (mSFKs) as key determinants of GF-induced NLRP3 inflammasome activation. Comprehensive analysis of the TKIs revealed that all TKIs we tested act as potent agonists for the NLRP3 inflammasome in human monocytic THP-1 cells and bone marrow-derived macrophages. Moreover, these TKIs share a common off-target activity against the mSFKs, such as c-Src, Fgr, and Fyn. Interestingly, loss of each kinase spontaneously stimulated the NLRP3 inflammasome activation in THP-1 cells. These results together suggest that NLRP3 senses hypoactivity of the mSFKs that is responsible for mitochondrial dysfunction. Thus, our findings demonstrate a mechanistic link between the NLRP3 inflammasome and mSFKs, which, to our knowledge, provides insights into a novel molecular basis and cellular function of the NLRP3 inflammasome.

Host determinants of TB disease as targets for HDTs development: A study protocol

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge. New diagnostic and therapeutic strategies are required to curb TB transmission. Here we outline a protocol to validate inflammatory proteins as potential biomarkers of TB disease and to evaluate the candidate genes as potential targets for host-directed therapy (HDT) development. Blood will be isolated from healthy, latent TB infected (LTBI) individuals and TB patients, and expression profiles of genes of interest will be determined using qPCR. A human monocytic cell line will be utilized to knock down genes of interest and to evaluate their contribution to Mtb infection. Pharmaceutical interception of target genes will be performed in peripheral blood mononuclear cells (PBMCs) infected with Mtb. This work will result in identification of TB associated inflammatory markers that can also be targeted for HDT development.

The Effects of Ginkgo biloba Extract on Autophagy in Human Macrophages Stimulated by Cigarette Smoke Extract.

To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

Combined Toxicity of the Most Common Indoor Aspergilli.

The most common Aspergilli isolated from indoor air samples from occupied buildings and a grain mill were extracted and analyzed for their combined (Flavi + Nigri, Versicolores + Nigri) cytotoxic, genotoxic and pro-inflammatory properties on human adenocarcinoma cells (A549) and monocytic leukemia cells induced in macrophages (THP-1 macrophages). Metabolite mixtures from the Aspergilli series Nigri increase the cytotoxic and genotoxic potency of Flavi extracts in A549 cells suggesting additive and/or synergistic effects, while antagonizing the cytotoxic potency of Versicolores extracts in THP-1 macrophages and genotoxicity in A549 cells. All tested combinations significantly decreased IL-5 and IL-17, while IL-1β, TNF-α and IL-6 relative concentrations were increased. Exploring the toxicity of extracted Aspergilli deepens the understanding of intersections and interspecies differences in events of chronic exposure to their inhalable mycoparticles.