Cytotoxicity In Human Macrophages Research Articles

Evaluating electronic cigarette cytotoxicity and inflammatory responses in vitro.

INTRODUCTIONCigarette smoking poses many health risks and can cause chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer of the lung and other organs. Smokers can substantially reduce their risks of these diseases by quitting, but nicotine addiction makes this difficult. Alternatives, such as electronic cigarettes (e-cigarettes), may provide a similar dose of nicotine, but expose users to fewer toxic chemicals than traditional cigarettes and may still be harmful especially for dual users, therefore, we sought to develop bioassays that can assess the potential toxicity and inflammatory response induced by e-cigarette liquids (e-liquids) with and without flavors.METHODSE-liquids with varying nicotine content and flavors were aerosolized through growth media and exposed to human bronchial epithelial cell line (BEAS-2B) and human monocyte-macrophage cell line (THP-1) in vitro. Cytotoxicity in response to e-cigarette aerosols was measured by MTT assay in BEAS-2B cells and inflammatory response was measured by TNF-α, IL-6, IL-8, and MCP-1 released from THP-1 cells. In addition, the oxidative stress marker, REDD1, and impact on phagocytosis, was assessed following exposure of BEAS-2B and THP-1 derived macrophages, respectively. Cigarette smoke extract was used as a positive control with known cytotoxicity and impairment of inflammatory response.RESULTSE-cigarette aerosols induced moderate cellular toxicity in bronchial epithelial cells. Our data also show that low nicotine levels are less damaging to the bronchial epithelial cells, and flavors in e-liquids influence the combined inflammatory response markers, phagocytosis, and REDD1 when examined in vitro.CONCLUSIONSOur in vitro bioassays can be utilized to effectively measure flavor and nicotine-induced effects of e-cigarettes on combined inflammatory response and cytotoxicity in human macrophages and human bronchial epithelial cells, respectively.

Cardiac glycosides cause cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis.

Cardiac glycosides inhibit Na+ /K+ -ATPase and are used to treat heart failure and arrhythmias. They can induce inflammasome activation and pyroptosis in macrophages, suggesting cytotoxicity, which remains to be elucidated in human tissues. To determine the cell-type specificity of this cytotoxicity, we used human monocyte-derived macrophages and non-adherent peripheral blood cells from healthy donors, plus omental white adipose tissue, stromal vascular fraction-derived pre-adipocytes and adipocytes from obese patients undergoing bariatric surgery. All these cells/tissues were treated with nanomolar concentrations of ouabain (50, 100, 500 nM) to investigate the level of cytotoxicity and the mechanisms leading to cell death. In white adipose tissue, we investigated ouabain-mediated cytotoxicity by measuring insulin sensitivity, adipose tissue function and extracellular matrix deposition ex vivo. Ouabain induced cell death through pyroptosis and apoptosis, and was more effective in monocyte-derived macrophages compared to non-adherent peripheral blood mononuclear cell populations. This cytotoxicity is dependent on K+ flux, as ouabain causes intracellular depletion of K+ and accumulation of Na+ and Ca2+ . Consistently, the cell death caused by these ion imbalances can be rescued by addition of potassium chloride to human monocyte-derived macrophages. Remarkably, when white adipose tissue explants from obese patients are cultured with nanomolar concentrations of ouabain, this causes depletion of macrophages, down-regulation of type VI collagen levels and amelioration of insulin sensitivity ex vivo. The use of nanomolar concentration of cardiac glycosides could be an attractive therapeutic treatment for metabolic syndrome, characterized by pathogenic infiltration and activation of macrophages. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.

MicroRNA-579 upregulation mediates death of human macrophages with mycobacterium tuberculosis infection

Mycobacterium tuberculosis (MTB) infection could induce death of host human macrophages, promoting bacterial spread. In the current study we tested the potential role of microRNA-579 (miR-579) in the death of macrophages infected with MTB. In the primary human macrophages MTB infection induced upregulation of miR-579 but downregulation of its mRNA targets, SIRT1 and PDK1, which were accompanied by significant macrophage death and apoptosis. miR-579 inhibition, by its anti-sense sequence, restored SIRT1-PDK1 expression and significantly attenuated MTB-induced cytotoxicity and apoptosis in human macrophages. Conversely, ectopic overexpression of miR-579 further downregulated SIRT1-PDK1 expression and exacerbated MTB-induced cytotoxicity in human macrophages. Further studies showed that cPWWP2A, the miR-579's endogenous sponge circRNA, was downregulated in MTB-infected macrophages. Conversely, forced overexpression of cPWWP2A, by a recombinant adeno-associated virus construct, reversed MTB-induced miR-579 upregulation and macrophage cytotoxicity. Taken together, our results show that miR-579 upregulation mediates MTB-induced macrophage cytotoxicity. Targeting cPWWP2A-miR-579 axis could be a novel strategy to protect human macrophages from MTB infection.

The effect of particle agglomeration on the formation of a surface-connected compartment induced by hydroxyapatite nanoparticles in human monocyte-derived macrophages

Agglomeration dramatically affects many aspects of nanoparticle–cell interactions. Here we show that hydroxyapatite nanoparticles formed large agglomerates in biological medium resulting in extensive particle uptake and dose-dependent cytotoxicity in human macrophages. Particle citration and/or the addition of the dispersant Darvan 7 dramatically reduced mean agglomerate sizes, the amount of particle uptake and concomitantly cytotoxicity. More surprisingly, agglomeration governed the mode of particle uptake. Agglomerates were sequestered within an extensive, interconnected membrane labyrinth open to the extracellular space. In spite of not being truly intracellular, imaging studies suggest particle degradation occurred within this surface-connected compartment (SCC). Agglomerate dispersion prevented the SCC from forming, but did not completely inhibit nanoparticle uptake by other mechanisms. The results of this study could be relevant to understanding particle–cell interactions during developmental mineral deposition, in ectopic calcification in disease, and during application of hydroxyapatite nanoparticle vectors in biomedicine.

The Role of NADPH Oxidase in Multi-Walled Carbon Nanotubes-Induced Oxidative Stress and Cytotoxicity in Human Macrophages

Recent studies suggest reactive oxygen species (ROS) induced in mammalian cells exposed to multi-walled carbon nanotubes (MWCNTs) could mediate the cytotoxicity. This study was conducted to determine the mechanisms responsible for MWCNTs-induced ROS production in human primary macrophages. Our results showed that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 100 microg/ml MWCNTs for 12 h. Concomitantly, MWCNTs induced membrane translocation of the NADPH oxidase subunits p47phox and p67phox, a signature event for NADPH oxidase activation. Pre-incubation with apocynin, a selective inhibitor of NADPH oxidase, prevented both membrane translocation of p47phox and superoxide production. Treatment with MWCNTs also resulted in an increased cytotoxicity in human primary macrophages that was significantly attenuated by both apocynin and antioxidants. These findings demonstrate that MWCNTs activate NADPH oxidase in human macrophages, which may contribute to ROS generation in MWCNTs treated-macrophages.

The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages

Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-α was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox™ or N-acetyl- l-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers.

Nox4 is a Novel Inducible Source of Reactive Oxygen Species in Monocytes and Macrophages and Mediates Oxidized Low Density Lipoprotein–Induced Macrophage Death

The enhanced formation of intracellular reactive oxygen species (ROS) induced by oxidized low-density lipoprotein (OxLDL) promotes macrophage death, a process likely to contribute to the formation of necrotic cores and the progression of atherosclerotic lesions. Yet macrophage deficiency of phagocytic NADPH oxidase (Nox2), the primary source of ROS in macrophages, does not reduce atherosclerotic lesion development in mice. This suggests an as yet unidentified NADPH oxidase may be present in macrophages and responsible for the intracellular ROS formation induced by OxLDL. The aim of this study was to identify the source of intracellular ROS involved in macrophage death. Nox4 was expressed in human monocytes and mature macrophages, and was localized to the endoplasmic reticulum and to defined foci within the nucleus. Nox4 colocalized with p22(phox), and both proteins were upregulated in response to OxLDL stimulation, whereas Nox2/gp91(phox) levels remained unchanged. Induction of Nox4 expression, intracellular ROS formation and macrophage cytotoxicity induced by OxLDL were blocked by MEK1/2 inhibition, but not by inhibitors of p38-MAPK (mitogen-activated protein kinase), JNK (Jun N-terminal kinase), or JAK2 (Janus kinase 2). Small interfering RNA knockdown of Nox4 inhibited both intracellular ROS production and macrophage cytotoxicity induced by OxLDL, whereas Nox4 overexpression enhanced both OxLDL-stimulated ROS formation and macrophage death. Nox4 is a novel source of intracellular ROS in human monocytes and macrophages. Induction of Nox4 by OxLDL is mediated by the MEK1/ERK pathway and required for OxLDL cytotoxicity in human macrophages, implicating monocytic Nox4 in atherogenesis.