Macrophage Secretome Research Articles

Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells.

Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combining both targeted- and immuno- therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying the effects of the macrophage secretome on cancer cells (on e.g. growth, drug response or gene expression) in co-cultures without direct cell contact interactions. This is an important aspect as it removes the presence of any phagocytic aspect to changes in the cancer cell number and behaviour. The in vitro THP-1 monocyte differentiation into polarized macrophages was used to study the effects of both M1 and M2 type populations of macrophages on melanoma cells (Smith et al., 2014; Tsuchiya et al., 1980). M1 type macrophages are classically thought to be tumour suppressing as opposed to M2 type macrophages, which are thought to possess tissue repairing and tumour growth promoting activities.

Abstract 12858: Left Ventricular Dysfunction Switches Cardiac Mesenchymal Stem/Stromal Cells Toward a Pro-Inflammatory Phenotype

Introduction and Hypothesis: Mesenchymal stem/stromal cells (MSCs) are appealing candidates for cardiac cell therapy due to their ease of preparation, immunoprivilege properties, reparative potential and safety. We aimed to test the hypothesis that the inflammatory environment following myocardial infarction (MI) and left ventricular (LV) dysfunction could modulate the reparative properties of resident and transplanted MSCs. Methods and Results: To test this hypothesis, we isolated and cultured resident cardiac and subcutaneous (SC) fat tissue MSCs from mice, 28 days after MI (LVEF: 27.9%±3.8) or sham operation (LVEF: 54.9%±3.8). Proliferation rate was highest in cardiac MSCs after MI, compared with other groups (p<0.001 after 96 hours). Next, we measured the levels of secreted cytokines in the medium collected from the different cells and found that MI polarized cardiac MSCs toward a pro-inflammatory profile and stimulated secretion of the inflammatory cytokines IL1α, IL1β, IL-12, IL-6 and TNF-α (p<0.05), as well as the monocyte recruitment factors: MIP-1α, RANTES and MCP-1 (p<0.05). In contrast, SC fat MSCs were less affected by MI and LV dysfunction and mainly decreased their macrophage recruitment and anti-inflammatory factors. Surprisingly, transplantation of cardiac and adipose tissue MSCs from both MI and sham operated animals, into mice hearts immediately after MI (n=5-8 per group), did not improve cardiac remodeling and function, compared with saline. To determine the mechanism behind MSC polarization, we stimulated cardiac MSCs from toll-like receptor 4 ( TLR4 ) deficient and wild-type mice, with the secretome of LPS-activated macrophages. Significantly, there was less inflammatory cytokine secretion in TLR4-deficient MSCs, compared with control suggesting that cardiac MSC polarization is mediated by TLR4 activation. Conclusions: Post MI inflammatory environment switches resident cardiac and implanted MSCs toward a pro-inflammatory phenotype and impair their reparative properties. This effect is mediated by TLR4 . Our findings are relevant to the pathogenesis and progression of LV remodeling, and could guide the selection and engineering of MSCs for heart repair.

Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders.

BackgroundAlthough major concerns exist regarding the potential consequences of human exposure to nanoparticles (NP), no human toxicological data is currently available. To address this issue, we took welders, who present various adverse respiratory outcomes, as a model population of occupational exposure to NP.The aim of this study was to evaluate if welding fume-issued NP could be responsible, at least partially, in the lung alterations observed in welders.MethodsA combination of imaging and material science techniques including ((scanning) transmission electron microscopy ((S)TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence (μXRF)), was used to characterize NP content in lung tissue from 21 welders and 21 matched control patients. Representative NP were synthesized, and their effects on macrophage inflammatory secretome and migration were evaluated, together with the effect of this macrophage inflammatory secretome on human lung primary fibroblasts differentiation.ResultsWelding-related NP (Fe, Mn, Cr oxides essentially) were identified in lung tissue sections from welders, in macrophages present in the alveolar lumen and in fibrous regions. In vitro macrophage exposure to representative NP (Fe2O3, Fe3O4, MnFe2O4 and CrOOH) induced the production of a pro-inflammatory secretome (increased production of CXCL-8, IL-1ß, TNF-α, CCL-2, −3, −4, and to a lesser extent IL-6, CCL-7 and −22), and all but Fe3O4 NP induce an increased migration of macrophages (Boyden chamber). There was no effect of NP-exposed macrophage secretome on human primary lung fibroblasts differentiation.ConclusionsAltogether, the data reported here strongly suggest that welding-related NP could be responsible, at least in part, for the pulmonary inflammation observed in welders. These results provide therefore the first evidence of a link between human exposure to NP and long-term pulmonary effects.

Amyloid-β Increases Metallo- and Cysteine Protease Activities in Human Macrophages

Background/Aims: Amyloid-β (Aβ) plays a crucial role in the onset and progression of atherosclerosis. Macrophages are a source of matrix metalloproteinases (MMPs), cysteine proteases and transforming growth factor (TGF)-β1 in the vascular wall. The aims of this study were to analyze the capacity of Aβ peptide (1-40) (Aβ40), Aβ peptide (1-42) (Aβ42) and fibrillar Aβ42 (fAβ42) to modulate the expression and activity of MMP-9, MMP-2 and tissue inhibitor of MMP-1 (TIMP-1) in human monocyte-derived macrophages (HMDM). Additionally, we analyzed whether Aβ internalization alters the secretion of cathepsin S (CatS) and TGF-β1 by macrophages. Methods: HMDM were exposed to native and fibrillar Aβ. MMPs and TIMP-1 expression was analyzed by real-time PCR, and MMP abundance by zymography. Protein levels of precursor and active forms of CatS were analyzed by Western blot and TGF-β1 levels by ELISA. Results: Aβ40, Aβ42 and especially fAβ42 strongly induced MMP-9/MMP-2 levels. Moreover, we showed enhanced active CatS and reduced TGF-β1 protein levels in the secretome of Aβ42 and fAβ42-exposed macrophages. Conclusions: Aβ can regulate the proinflammatory state of human macrophages by inducing metallo- and cysteine protease levels and by reducing TGF-β1 secretion. These effects may be crucial in atherosclerosis progression.

Macrophage Subpopulations Are Essential for Infarct Repair With and Without Stem Cell Therapy

This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages. The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood. We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging. The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80(+)CD206(+)) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy. Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair.

Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages

Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense.

Characterization of the Placental Macrophage Secretome: Implications for Antiviral Activity

It is well documented that placental macrophages show lower levels of HIV-1 infection than monocyte-derived macrophages (MDM). We used proteomic methods to test the hypothesis that placental macrophages secrete different proteins as compared to MDM that may contribute to decreased HIV-1 replication. Placental macrophages and MDM were cultured for 12 days and supernatant was collected. To characterize supernatants, the protein profiles of placental macrophages and MDM were compared using the protein chip assay. Subsequently, proteins were separated by one-dimensional gel electrophoresis and identified by tandem mass spectrometry at the corresponding mass to charge (m/z) range of 5000–20,000. Significant differences were found between placental macrophages and MDM in seven protein peaks with m/z values of 6075, 6227, 11,662, 14,547, 6158, 7740, and 11,934 on the CM10 and IMAC chips. After sequencing and identification, five proteins were validated for differential expression in placental macrophages and MDM by Western blot analyses. Peroxiredoxin 5, found to be more abundant in placental macrophage supernatants, is important in the cellular antioxidant mechanisms, and other members of its family have shown antiviral activity. Cystatin B was less abundant in PM supernatant, and decreased intracellular levels have recently been shown to be associated with lower HIV-1 replication in placental macrophages than in MDM. This study elucidates for the first time the placental macrophage secretome corresponding to 5000–20,000 Da and advances our understanding of the proteins secreted in the placenta that can protect the fetus against HIV-1 and other viral infections.

Investigating the human immunodeficiency virus type 1-infected monocyte-derived macrophage secretome

Mononuclear phagocytes (bone marrow monocyte-derived macrophages, alveolar macrophages, perivascular macrophages, and microglia) are reservoirs and vehicles of dissemination for the human immunodeficiency virus type-1 (HIV-1). How virus alters mononuclear phagocyte immunoregulatory activities to complete its life cycle and influence disease is incompletely understood. In attempts to better understanding the influence of virus on macrophage functions, we used one-dimensional electrophoresis, and liquid chromatography tandem mass spectrometry to analyze the secretome of HIV-1-infected human monocyte-derived macrophages. We identified 110 proteins in culture supernatants of control (uninfected) and virus-infected cells. Differentially expressed cytoskeletal, enzymes, redox, and immunoregulatory protein classes were discovered and validated by Western blot tests. These included, but were not limited to, cystatin C, cystatin B, chitinase 3-like 1 protein, cofilin-1, l-plastin, superoxide dismutase, leukotriene A 4 hydrolase, and α-enolase. This study, using a unique proteomics platform, provides novel insights into virus–host cell interactions that likely affect the functional role of macrophages in HIV disease.

Two-dimensional maps and databases of the human macrophage proteome and secretome.

Macrophages exert a crucial, but still incompletely known, role in complex disorders such inflammatory, immunological, and infectious diseases. A differential proteomic approach should help to elucidate the macrophage dysfunctions involved in these diseases. With this goal in mind, we established the first two-dimensional maps of the human macrophage proteome and secretome. Intracellular and secreted proteins were extracted from monocyte-derived macrophages obtained from healthy donors (n = 16), and separated by two-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two patterns specific of the macrophage proteome and secretome, respectively. A total of 127 and 66 distinct intracellular and secreted polypeptide spots, corresponding to 100 and 38 different proteins, respectively, were identified by matrix assisted laser desorption/ionisation-mass spectrometry. The two-dimensional reference maps and databases resulting from this study confirm that macrophages are involved in a wide range of biological functions, and that they provide a useful tool for a wide array of investigators involved in macrophage biology, allowing to investigate the macrophage protein changes associated with various disorders or environmental stimuli.