Human Monocytic Leukemia Cell Research Articles

Immunomodulatory role of non-ionizing electromagnetic radiation in human leukemia monocytic cell line

In daily life, people are usually exposed to radiofrequency radiations (RFR). The effects of RFR on human physiology have been a major source of controversy since the WHO declared that these radiations are a type of environmental energy that interacts with the physiological functioning of the human body. The immune system provides internal protection and promotes long-term health and survival. However, the relevant research on the innate immune system and radiofrequency radiation is scant. In this connection, we hypothesized that innate immune responses would be influenced by exposure to non-ionizing electromagnetic radiation from mobile phones in a cell-specific and time-dependent manner. To test this hypothesis, human leukemia monocytic cell lines were exposed to 2318 MHz (MHz) RFR emitted by mobile phones at a power density of 0.224 W/m2 in a controlled manner for various time durations (15, 30, 45, 60, 90, and 120 min). Systematic studies on cell viability, nitric oxide (NO), superoxide (SO), pro-inflammatory cytokine production, and phagocytic assays were performed after the irradiation. The duration of exposure seems to have a substantial influence on the RFR-induced effects. It was noticed that after 30 min of exposure, the RFR dramatically enhanced the pro-inflammatory cytokine IL-1α level as well as reactive species such as NO and SO generation as compared to the control. In contrast, the RFR dramatically reduced the phagocytic activity of monocytes during 60 min of treatment when compared to the control. Interestingly, the irradiated cells restored their normal functioning until the final 120-min of exposure. Furthermore, mobile phone exposure had no influence on cell viability or TNF-α level. The results showed that RFR exhibits a time-dependent immune-modulatory role in the human leukemia monocytic cell line. Nevertheless, more research is needed to further determine the long-term effects and precise mechanism of action of RFR.

Itraconazole Modulates Phospholipid Levels in Tumor-associated Macrophages.

Itraconazole, an antifungal drug, repolarizes pro-tumorigenic M2 tumor-associated macrophages to anti-tumorigenic M1-like phenotypes, thereby inhibiting the proliferation of cancer cells; however, the underlying mechanism remains unclear. Therefore, we investigated the effect of itraconazole on membrane-associated lipids in tumor-associated macrophages (TAM). M1 and M2 macrophages were derived from the human monocyte leukemia cell line (THP-1) and cultured with or without 10 μM itraconazole. Cells were homogenized and subjected to liquid chromatography/mass spectrometry (LC/MS) analysis to estimate the glycerophospholipid levels in the cells. Lipidomic analysis results, displayed on a volcano plot, revealed that itraconazole-induced altered phospholipid composition, with more pronounced changes in M2 macrophages than in M1. Notably, itraconazole significantly increased intracellular phosphatidylinositol and lysophosphatidylcholine levels in M2 macrophages. Itraconazole modulates the lipid metabolism of TAMs, which could have implications for the development of novel cancer therapies.

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

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.

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.

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.

Amnion-derived serum amyloid A1 participates in sterile inflammation of fetal membranes at parturition.

Sterile inflammation of fetal membranes is an indispensable event of normal parturition. However, triggers of sterile inflammation are not fully resolved. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver. Fetal membranes can also synthesize SAA1 but its functions are not well defined. Given the role of SAA1 in the acute phase response to inflammation, we postulated that SAA1 synthesized in the fetal membranes may be a trigger of local inflammation at parturition. The changes of SAA1 abundance in parturition were studied in the amnion of human fetal membranes. The role of SAA1 in chemokine expression and leukocyte chemotaxis was examined in cultured human amnion tissue explants as well as primary human amnion fibroblasts. The effects of SAA1 on monocytes, macrophages and dendritic cells were investigated in cells derived from a human leukemia monocytic cell line (THP-1). SAA1 synthesis increased significantly in human amnion at parturition. SAA1 evoked multiple chemotaxis pathways in human amnion fibroblasts along with upregulation of a series of chemokines via both toll-like receptor 4 (TLR4) and formyl peptide receptor 2 (FPR2). Moreover, SAA1-conditioned medium of cultured amnion fibroblasts was capable of chemoattracting virtually all types of mononuclear leukocytes, particularly monocytes and dendritic cells, which reconciled with the chemotactic activity of conditioned medium of cultured amnion tissue explants collected from spontaneous labor. Furthermore, SAA1 could induce the expression of genes associated with inflammation and extracellular matrix remodeling in monocytes, macrophages and dendritic cells derived from THP-1. SAA1 is a trigger of sterile inflammation of the fetal membranes at parturition.

Itraconazole Repolarizes Tumor-associated Macrophages and Suppresses Cervical Cancer Cell Growth.

Itraconazole (ITZ), an antifungal agent, has been reported to have anti-tumor effects in patients with multiple cancer types. We investigated the involvement of tumor-associated macrophages (TAMs) in its tumor-agnostic mechanism. M1 and M2 macrophages were established from human monocyte leukemia cell line (THP-1) and their phenotypes were determined morphologically. Cell membrane antigens and secreted proteins were evaluated by western blots and enzyme-linked immunosorbent assay, respectively. The proteomic profiling of cells was done by liquid chromatography with tandem mass spectrometry and analyzed. Viability of cervical cancer cells (CaSki) was evaluated after addition of the supernatant of M2 macrophages and during co-culture with M2 macrophages, with or without 10-5 M ITZ. Co-culture of M1 macrophages inhibited the proliferation of CaSki cells (p=0.012), while that of M2 macrophages promoted their proliferation (p<0.0001). After treatment of M2 macrophages with ITZ for 24 h, they changed into M1-like shape with decreased expression of cluster of differentiation 163 (CD163) and chemokine ligand 18 (CCL18). The M1-like shape was maintained for 7 weeks of ITZ treatment and reverted to original after ITZ removal. Proteomic analysis of ITZ treated-M2 macrophages also demonstrated M1-like signature including the elevated levels of tumor necrosis factor (TNF)-related proteins. After treatment with ITZ, both the supernatant of the M2 macrophages and the co-culture with M2 macrophages significantly inhibited the proliferation of CaSki cells (each, p<0.0001). ITZ repolarized M2 macrophages to M1 type and suppressed cervical cancer cell growth demonstrating TAM-mediated anti-cancer activity of ITZ.

Specific microRNA Signature Kinetics in Porphyromonas gingivalis-Induced Periodontitis.

Porphyromonas gingivalis is one of the major bacteria constituting the subgingival pathogenic polymicrobial milieu during periodontitis. Our objective is to determine the global microRNA (miRNA, miR) expression kinetics in 8- and 16-weeks duration of P. gingivalis infection in C57BL/6J mice and to identify the miRNA signatures at specific time-points in mice. We evaluated differential expression (DE) miRNAs in mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels. The bacterial colonization, alveolar bone resorption (ABR), serum immunoglobulin G (IgG) antibodies, and bacterial dissemination were confirmed. In addition, all the infected mice showed bacterial colonization on the gingival surface, significant increases in ABR (p < 0.0001), and specific IgG antibody responses (p < 0.05-0.001). The miRNA profiling showed 26 upregulated miRNAs (e.g., miR-804, miR-690) and 14 downregulated miRNAs (e.g., miR-1902, miR-1937a) during an 8-weeks infection, whereas 7 upregulated miRNAs (e.g., miR-145, miR-195) and one downregulated miR-302b were identified during a 16-weeks infection. Both miR-103 and miR-30d were commonly upregulated at both time-points, and all the DE miRNAs were unique to the specific time-points. However, miR-31, miR-125b, miR-15a, and miR-195 observed in P. gingivalis-infected mouse mandibles were also identified in the gingival tissues of periodontitis patients. None of the previously identified miRNAs reported in in vitro studies using cell lines (periodontal ligament cells, gingival epithelial cells, human leukemia monocytic cell line (THP-1), and B cells) exposed to P. gingivalis lipopolysaccharide were observed in the in vivo study. Most of the pathways (endocytosis, bacterial invasion, and FcR-mediated phagocytosis) targeted by the DE miRNAs were linked with bacterial pathogen recognition and clearance. Further, eighteen miRNAs were closely associated with the bacterial invasion of epithelial cells. This study highlights the altered expression of miRNA in gingiva, and their expression depends on the time-points of infection. This is the first in vivo study that identified specific signature miRNAs (miR-103 and miR-30d) in P. gingivalis invasion of epithelial cells, establishes a link between miRNA and development of periodontitis and helping to better understand the pathobiology of periodontitis.

An optical pH-sensor integrated microfluidic platform multilayered with bacterial cellulose and gelatin methacrylate to mimic drug-induced lung injury

Drug-induced lung injury leading to pulmonary fibrosis is an interstitial lung pathology destructing the architecture of lung. Although animal models have been used to understand the progression of the pathology, it is often difficult to correlate with human physiology and the readouts rely on end-point analyzes. Herein, an optical pH-sensor integrated biomimetic microfluidic model was designed to simulate the drug-induced lung injury along with the cytotoxicity related acidity changes of the cell culture media. In the model, interstitial region was adopted via bacterial cellulose-based vascular membrane below the connective tissue, which was emulated with hydrogel-based gelatin-methacrylate (GelMA). During 12 days of dynamic culture, the increased levels of transforming growth factor-1 (TGF-β1) and lactate dehydrogenase (LDH) are observed confirming the real-time pH changes due to the profibrotic features. Recruitment of human leukemia monocytic (THP-1) cells caused by fatal epithelial/fibroblast cross-talk was observed as an anti-inflammatory response with the effect of cytotoxicity and profibrotic TGF-β1. Moreover, decreased collagen type I secretion levels following the anti-fibrotic drug provided evidence for the model reliability.

The Monocytic Cell Line THP-1 as a Validated and Robust Surrogate Model for Human Dendritic Cells.

We have implemented an improved, cost-effective, and highly reproducible protocol for a simple and rapid differentiation of the human leukemia monocytic cell line THP-1 into surrogates for immature dendritic cells (iDCs) or mature dendritic cells (mDCs). The successful differentiation of THP-1 cells into iDCs was determined by high numbers of cells expressing the DC activation markers CD54 (88%) and CD86 (61%), and the absence of the maturation marker CD83. The THP-1-derived mDCs are characterized by high numbers of cells expressing CD54 (99%), CD86 (73%), and the phagocytosis marker CD11b (49%) and, in contrast to THP-1-derived iDCs, CD83 (35%) and the migration marker CXCR4 (70%). Treatment of iDCs with sensitizers, such as NiSO4 and DNCB, led to high expression of CD54 (97%/98%; GMFI, 3.0/3.2-fold induction) and CD86 (64%/96%; GMFI, 4.3/3.2-fold induction) compared to undifferentiated sensitizer-treated THP-1 (CD54, 98%/98%; CD86, 55%/96%). Thus, our iDCs are highly suitable for toxicological studies identifying potential sensitizing or inflammatory compounds. Furthermore, the expression of CD11b, CD83, and CXCR4 on our iDC and mDC surrogates could allow studies investigating the molecular mechanisms of dendritic cell maturation, phagocytosis, migration, and their use as therapeutic targets in various disorders, such as sensitization, inflammation, and cancer.

In vitro cytotoxicity of Mn(I) and Ru(II) carbonyls with a diphenyl pyridyl phosphine coligand towards leukaemia.

Human acute monocytic leukaemia cells were tested under both dark and light conditions for their susceptibility to Mn(I) and Ru(II) carbonyl complexes with a diphenyl pyridyl phosphine coligand. The Ru(II) complex (IC50 = 7.13 ± 0.8 μM) displayed higher outstanding potency against leukaemia than the Mn(I) analogue (54.58 ± 4.1 μM) in the dark and both complexes were completely harmless to healthy mouse bone marrow cells.

Role of Serum C-C Motif Chemokine Ligand 2 Monocyte Chemotactic Activities in Patients with Non Small Cell Lung Cancer

To examine C-C motif chemokine ligand 2 chemotactic activities, expression and clinical relevance in the peripheral serum of non-small cell lung cancer patients is the objective of the study. The non-small cell lung cancer group consisted of 128 patients diagnosed with the disease in our institution between January 2018 and March 2022, while the healthy group consisted of 112 individuals. By using an enzyme-linked immunosorbent assay, the two group’s peripheral serum was examined for C-C motif chemokine ligand 2 expression levels. The monocyte chemotactic activity of blood plasma from the two groups was assessed using the Transwell chamber technique after human monocytic leukaemia cell line THP-1 cells were grown. Analysis was done on C-C motif chemokine ligand 2 expression levels and the relationship between monocyte chemotactic activity and histopathological features in non-small cell lung cancer patients. Age and gender did not significantly connect with the expression level of C-C motif chemokine ligand 2 or its monocyte chemotactic activity (p>0.05); however, smoking, pathological categorization, tumor, nodes and metastases stage, tumor stage, nodes stage and tumor diameter did (p<0.05). The expression level of C-C motif chemokine ligand 2 was 235.4±25.7 pg/ml in the peripheral serum of non-small cell lung cancer patients and 45.7±10.5 pg/ml in the healthy population; the monocyte chemotactic activity of non-small cell lung cancer patients was 236.5 % and that of healthy people was 86.5 %. They differed from one other in a statistically significant way (p<0.05). In comparison to the normal control group (r=0.87), the lung cancer group’s correlation coefficient between blood C-C motif chemokine ligand 2 and monocyte chemotactic activity was considerably lower (r=0.39). Patients with non-small cell lung cancer patients exhibit inadequate monocyte chemotactic activity, which may one day be targeted therapeutically.

Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite.In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1β), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1β, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Differential roles of ApoE isoforms in regulation of inflammatory cytokine expression

AbstractBackgroundApoE Ɛ4 has long been known as a top genetic risk factor in the development of late‐onset Alzheimer’s disease (LOAD). More recently the genetic variants of the innate immune receptor TREM2 such as R47H, which confers a similar risk as ApoE4 in LOAD, have been identified as genetic risk factors in the development of many neurodegenerative diseases with dysregulated neuroinflammation. TREM2 is expressed highly on microglia and macrophages and plays key roles in inflammation regulation. Our previous study (Dorey et al. 2017) has shown that ApoE isoforms play differential roles in Aβ‐induced inflammatory response. Atagi et al (2015) identified that ApoE is a ligand for TREM2. Given the physiological relationship between ApoE and TREM2 while also considering the three isoforms of ApoE, it is crucial to investigate and understand the mechanisms underlying the possibility differential ApoE isoform signaling through TREM2 surface receptor.MethodTHP‐1 (human monocytic leukemia cell line) cells were differentiated into macrophages (M0) using PMA and subsequently polarized to M1 and M2 using IFN‐γ/LPS and IL‐4/IL‐13, respectively. Following differentiation, 3uM of ApoE2, E3, or E4 was added to polarization media. RNA was extracted following 2 day polarization and used for RT‐qPCR in relative gene expression quantification.ResultApoE2 strongly stimulated the expression of anti‐inflammatory cytokines IL‐10 and TARC/CCL17 in M1 and M2 macrophages as compared to control, ApoE3 and ApoE4. While ApoE4 showed a massive reduction in these same transcripts in M0 and M2 macrophages. In contrast, ApoE4 strongly promoted the expression of pro‐inflammatory cytokines IL‐1β and TNF‐α in M0, M1, and M2 macrophages as compared to control, ApoE3 and ApoE2.ConclusionThe ability for ApoE2 to promote the expression of anti‐inflammatory cytokines in macrophages, suggests that ApoE2 may play a neuroprotective role; while ApoE4’s ability to promote inflammatory cytokine expression indicates its pro‐inflammatory role. Both ApoE2 and ApoE4 may play their roles via interacting with TREM2.

Trifluoromethylcinnamanilide Michael Acceptors for Treatment of Resistant Bacterial Infections

A series of thirty-two anilides of 3-(trifluoromethyl)cinnamic acid (series 1) and 4-(trifluoromethyl)cinnamic acid (series 2) was prepared by microwave-assisted synthesis. All the compounds were tested 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 compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. (2E)-3-[3-(Trifluoromethyl)phenyl]-N-[4-(trifluoromethyl)phenyl]prop-2-enamide (1j), (2E)-N-(3,5-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]prop-2-enamide (1o) and (2E)-N-[3-(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)-phenyl]prop-2-enamide (2i), (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)phenyl]-prop-2-enamide (2p) showed antistaphylococcal (MICs/MBCs 0.15-5.57 µM) as well as anti-enterococcal (MICs/MBCs 2.34-44.5 µM) activity. The growth of M. marinum was strongly inhibited by compounds 1j and 2p in a MIC range from 0.29 to 2.34 µM, while all the agents of series 1 showed activity against M. smegnatis (MICs ranged from 9.36 to 51.7 µM). The performed docking study demonstrated the ability of the compounds to bind to the active site of the mycobacterial enzyme InhA. The compounds had a significant effect on the inhibition of bacterial respiration, as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity but also bactericidal activity. Preliminary in vitro cytotoxicity screening was assessed using the human monocytic leukemia cell line THP-1 and, except for compound 2p, all effective agents did show insignificant cytotoxic effect. Compound 2p is an interesting anti-invasive agent with dual (cytotoxic and antibacterial) activity, while compounds 1j and 1o are the most interesting purely antibacterial compounds within the prepared molecules.

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n=49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Post-prostatic-massage urine exosomes of men with chronic prostatitis/chronic pelvic pain syndrome carry prostate-cancer-typical microRNAs and activate proto-oncogenes.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a high prevalence of up to 15% and accounts for 90-95% of prostatitis diagnoses, and yet its etiopathogenesis and link to prostate cancer (PCa) are still unclear. Here, we investigated microRNAs in exosomes isolated from blood and post-prostatic-massage urine of CP/CPPS type IIIb patients and healthy men. THP-1 monocytes (human leukemia monocytic cell line) were treated with exosomes and subjected to mRNA arrays "Cancer Inflammation and Immunity Crosstalk" and "Transcription Factors." Using The Cancer Genome Atlas, the expression of CP/CPPS-associated microRNAs was analyzed in PCa and normal prostate tissue. In silico functional studies were carried out to explore the disease ontology of CP/CPPS. In CP/CPPS, urine exosomes exhibited significant upregulation of eight PCa-specific microRNAs (e.g., hsa-miR-501, hsa-miR-20a, and hsa-miR-106), whose target genes were significantly enriched for GO terms, hallmark gene sets, and pathways specific for carcinogenesis. In THP-1 monocytes, CP/CPPS-derived urine exosomes induced upregulation of PCa-associated proinflammatory genes (e.g., CCR2 and TLR2) and proto-oncogene transcription factors (e.g., MYB and JUNB). In contrast, CP/CPPS-derived blood exosomes exhibited molecular properties similar to those of healthy men. Thus, CP/CPPS exhibits molecular changes that constitute a risk for PCa and should be considered in the development of PCa biomarkers and cancer screening programs.

Whey protein hydrolysate mitigates both inflammation and endotoxin tolerance in THP-1 human monocytic leukemia cells.

It is important to control both inflammation and immunosuppression after severe insults, such as sepsis, trauma, and surgery. Endotoxin tolerance is one of the immunosuppressive conditions and it has been known that endotoxin tolerance relates to poorer clinical outcomes in patients with severe insults. This study investigated whether whey protein hydrolysate (WPH) mitigates inflammation and endotoxin tolerance in THP-1 human monocytic leukemia cells. Endotoxin tolerance can be experimentally reproduced by two consecutive stimulations with lipopolysaccharide (LPS). THP-1 cells were incubated with LPS and WPH (first stimulation). After collecting the culture supernatant to evaluate the effect on inflammation, the cells were washed and restimulated by 100 ng/ml LPS (second stimulation). The culture supernatant was again collected to evaluate the effect on endotoxin tolerance. Concentrations of LPS and WPH in the first stimulation were adjusted to evaluate their dose dependency. Cytokine levels in the supernatant were determined by enzyme-linked immunosorbent assay. Statistical analysis was performed using the student's t-test or Dunnett's test. Five mg/ml WPH significantly decreased interleukin (IL)-6 (p = .006) and IL-10 (p < .001) levels after the first LPS stimulation (1000 ng/ml). WPH significantly increased tumor necrosis factor-alpha (p < .001) and IL-10 (p = .014) levels after the second LPS stimulation. The suppressive effect of WPH on inflammation and endotoxin tolerance was dependent on the concentrations of LPS and WPH. The effective dose of WPH for endotoxin tolerance was lower than its effective dose for inflammation. WPH mitigated both inflammation and endotoxin tolerance. Therefore, WPH might be a candidate for valuable food ingredients to control both inflammation and immunosuppression after severe insults.