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Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.

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In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points.

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Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host-EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts.

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Background: Pesticides are indispensable for the cultivation of crops, especially those of economic importance, such as soybeans. Data on the annual use of herbicides in crops show that they correspond to 50%, making it the most used in agriculture. Aim: Therefore, the aim of this study was to evaluate the toxicity of the three commercial herbicides (clomazone, glyphosate, and sulfentrazone) in THP-1 cells. Methods: Cells were incubated with 0-5,000 mg/L of the herbicides for 24 h at 37 °C for cytotoxicity evaluation. Additionally, a few toxicological pathways such as reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of pesticides, were also evaluated. Results: A potential immunotoxic effect of the herbicides on THP-1 cells was observed, especially glyphosate, as it is a powerful agent of cellular immunotoxicity. It was also possible to verify an increase in oxidative stress and IL-8 levels and mitochondrial dysfunction. Conclusion: All herbicides showed cytotoxic effects in THP-1 monocytes, which were related to mitochondrial impairment.

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Objective: This present work aimed to evaluate the activation of NF-kB in human monocyte THP-1 transfected with GFP cell line (THP-1-NFkB-GFP) aiming to evaluate the action of snake toxins on cell activation. Methods: To carry out this work, it was necessary to cultivate THP-1 and THP-1-NFkB-GFP cell lines in supplemented RPMI medium, Bothropstoxin-I and Bothropstoxin-II (BthTX-I and BthTX-II) isolation from Bothrops jararacussu venom by cation exchange chromatography analysis of the purity of the toxins carried out using the SDS-PAGE method, an analysis of the cytotoxic activity of BthTX-I and BthTX-II in THP-1-NFkB-GFP cell line using MTT method, and THP-1-NFkB-GFP cell activation, using LPS followed analysis by confocal microscope. Results: The toxins were successufully purifield to use in subsequent experiments. In citotoxicity assays it was verified that the toxins in the concentrations used did not induce the THP-1 death. Furthermore, the incubation of THP-1-NFkB-GFP with LPS was able to induce cell activation and GFP expression. Conclusion: In conclusion, THP-1 cells are activated via the NF-kB pathway by lipopolysaccharide (LPS).

Objetivo: Este presente trabalho tem como propósito avaliar a ativação do NF-kB em monócitos humanos da linhagem THP-1 transfectado com a proteína GFP (THP-1- NFkB-GFP) para posterior estudo da ação de toxinas de serpentes sobre a ativação celular. Métodos: Para realização deste trabalho foram realizados o cultivo das linhagens de monócitos humanos THP-1 e THP-1-GFP em meio RPMI suplementado, a purificação de Bothropstoxina-I e Bothropstoxina-II (BthTX-I e BthTX-II) do veneno de Bothrops jararacussu pelo método de cromatografia de troca catiônica, confirmação da pureza das toxinas pelo método de SDS-PAGE, a análise da atividade citotóxica de BthTX-I e BthTX-II nas células THP-1-GFP pelo método de MTT, e ensaios de ativação das células THP-1-NFkB-GFP analisados em microscópio confocal. Resultados: As toxinas foram obtidas em pureza a quantidade para a realização dos experimentos. Nos ensaios de citotoxicidade foi determinado que as toxinas nas concentrações utilizadas não induziram a morte das células THP-1. Além disso, a incubação das células com LPS foi capaz ativar as células THP-1-NFkB-GFP expressar a GFP. Conclusão: Em conclusão as células THP-1 são ativadas pela via NF-kB por lipopolissacarídeo (LPS).

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Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

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Acute monocytic leukemia is a type of myeloid leukemia that develops in monocytes. The current clinical therapies for leukemia are unsatisfactory due to their side effects and nonspecificity toward target cells. Some lectins display antitumor activity and may specifically recognize cancer cells by binding to carbohydrate structures on their surface. Therefore, this study evaluated the response of the human monocytic leukemia cell lines THP-1 to the Olneya tesota PF2 lectin. The induction of apoptosis and reactive oxygen species production in PF2-treated cells was evaluated by flow cytometry, and the lectin-THP-1 cell interaction and mitochondrial membrane potential were evaluated by confocal fluorescence microscopy. PF2 genotoxicity was evaluated by DNA fragmentation analysis via gel electrophoresis. The results showed that PF2 binds to THP-1 cells, triggers apoptosis and DNA degradation, changes the mitochondrial membrane potential, and increases reactive oxygen species levels in PF2-treated THP-1 cells. These results suggest the potential use of PF2 for developing alternative anticancer treatments with enhanced specificity.

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One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

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Statins are currently the treatment of choice for hypercholesterolemia. However, wide interindividual variability has been observed in the response to treatment. Recent studies have reported the role of lncRNAs in the metabolism of lipids; nevertheless, there are few studies to date that show their role in the response to treatment with statins. Thus, the aim of this study was to assess the levels of expression of three lncRNAs (RP1-13D10.2; MANTIS; lncHR1) associated with genes involved in cholesterol homeostasis in leukocyte cells of hypercholesterolemic patients after treatment with atorvastatin and compare them with levels in subjects with normal cholesterol levels. A secondary aim was to assess the levels of expression in monocytic THP-1 cells differentiated to macrophages. The study included 20 subjects with normal cholesterol (NC) levels and 20 individuals with hypercholesterolemia (HC). The HC patients were treated with atorvastatin (20 mg/day/4 weeks). THP-1 cells were differentiated to macrophages with PMA and treated with different doses of atorvastatin for 24 h. Expression of lncRNAs was determined by RT-qPCR. The lncRNAs RP1-13D10.2 (p < 0.0001), MANTIS (p = 0.0013) and lncHR1 (p < 0.0001) presented increased expression in HC subjects compared with NC subjects. Furthermore, atorvastatin had a negative regulatory effect on the expression of lncHR1 (p < 0.0001) in HC subjects after treatment. In vitro, all the lncRNAs showed significant differences in expression after atorvastatin treatment. Our findings show that the lncRNAs tested present differential expression in HC patients and play a role in the variability reported in the response to atorvastatin treatment. Further research is needed to clarify the biological impact of these lncRNAs on cholesterol homeostasis and treatment with statins.