Search

Mesoporous silica-based particles are promising candidates for biomedical applications. Here, we address the importance of macrophage activation status for internalization of AMS6 (approx. 200 nm in diameter) versus AMS8 (approx. 2 μm) mesoporous silica particles and the role of different phagocytosis receptors for particle uptake. To this end, FITC-conjugated silica particles were used. AMS8 were found to be non-cytotoxic both for M-CSF-stimulated (anti-inflammatory) and GM-CSF-stimulated (pro-inflammatory) macrophages, whereas AMS6 exhibited cytotoxicity towards M-CSF-stimulated, but not GM-CSF-stimulated macrophages; this toxicity was, however, mitigated in the presence of serum. AMS8 triggered the secretion of pro-inflammatory cytokines in M-CSF-activated cells. Class A scavenger receptor (SR-A) expression was noted in both M-CSF and GM-CSF-stimulated macrophages, although the expression was higher in the former case, and gene silencing of SR-A resulted in a decreased uptake of AMS6 in the absence of serum. GM-CSF-stimulated macrophages expressed higher levels of the mannose receptor CD206 compared to M-CSF-stimulated cells, and uptake of AMS6, but not AMS8, was reduced following the downregulation of CD206 in GM-CSF-stimulated cells; particle uptake was also suppressed by mannan, a competitive ligand. These studies demonstrate that macrophage activation status is an important determinant of particle uptake and provide evidence for a role of different macrophage receptors for cell uptake of silica particles., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.

Sepsis is a major cause of death for hospitalized patients and is characterized by massive overreaction of immune responses to invading pathogens which is mediated by cytokines. For decades, there has been no effective treatment for sepsis. Sialic acid-binding, Ig-like lectin-9 (Siglec-9), is an immunomodulatory receptor expressed primarily on hematopoietic cells which is involved in various aspects of inflammatory responses and is a potential target for treatment of sepsis. The aim of the present study was to develop a human anti-Siglec-9 Fab fragment, which was named hS9-Fab03 and investigate its immune activity in human macrophages. We began by constructing the hS9-Fab03 prokaryotic expression vector from human antibody library and phage display. Then, we utilized a multitude of assays, including SDS-PAGE, Western blotting, ELISA, affinity, and kinetics assay to evaluate the binding affinity and specificity of hS9-Fab03. Results demonstrated that hS9-Fab03 specifically bind to Siglec-9 antigen with high affinity, and pretreatment with hS9-Fab03 could attenuate lipopolysaccharide (LPS)-induced TNF-α, IL-6, IL-1β, IL-8, and IFN-β production in human PBMC-derived macrophages, but slightly increased IL-10 production in an early time point. We also observed similar results in human THP-1-differentiated macrophages. Collectively, we prepared the hS9-Fab03 with efficient activity for blocking LPS-induced pro-inflammatory cytokines production in human macrophages. These results indicated that ligation of Siglec-9 with hS9-Fab03 might be a novel anti-inflammatory therapeutic strategy for sepsis.

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the ΔpdpC and ΔiglG mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both ΔpdpC and ΔiglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The ΔpdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the ΔiglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The ΔiglG mutant, with its unique phenotype, is a potential vaccine candidate., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Tuberculosis (TB) is a serious disease that is characterized by Mycobacterium tuberculosis (M.tb)-triggered immune system impairment and lung tissue damage shows limited treatment options. MicroRNAs (miRNAs) are regulators of gene expression that play critical roles in many human diseases, and can be up- or downregulated by M.tb infection in macrophage. Recently, tissue inhibitor of matrix metalloproteinase (TIMP) 3 has been found to play roles in regulating macrophage inflammation. Here, we found that TIMP3 expression was regulated by miR-206 in M.tb-infected THP-1 human macrophages. In THP-1 cells infected with M.tb, the miR-206 level was significantly upregulated and the expression of TIMP3 was markedly decreased when the secretion of inflammatory cytokines was increased. Inhibition of miR-206 markedly suppressed inflammatory cytokine secretion and upregulated the expression of TIMP3. In contrast, the upregulation of miR-206 promoted the matrix metalloproteinase (MMP) 9 levels and inhibited TIMP3 levels. Using a dual-luciferase reporter assay, a direct interaction between miR-206 and the 3'-untranslated region (UTR) of TIMP3 was confirmed. SiTIMP3, the small interfering RNA (siRNA) specific for TIMP3, significantly attenuated the suppressive effects of miR-206-inhibitor on inflammatory cytokine secretion and MMP9 expression. Our data suggest that miR-206 may function as an inflammatory regulator and drive the expression of MMP9 in M.tb-infected THP-1 cells by targeting TIMP3, indicating that miR-206 is a potential therapeutic target for patients with TB., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Type D personality (Type D) is an independent psychosocial risk factor for poor cardiac prognosis and increased mortality in patients with cardiovascular disease (CVD), but the involved mechanisms are poorly understood. Macrophages play a pivotal role in atherosclerosis, the process underlying coronary artery disease (CAD). We investigated macrophage superoxide anion production in production in CAD patients with and without Type D., Methods and Results: We studied 20 male CAD patients with Type D (M:66.7±9.9years) and 20 age-matched male CAD patients without Type D (M:67.7±8.5years). Type D was measured using the DS14 questionnaire with the two subscales 'negative affectivity' and 'social inhibition'. We assessed macrophage superoxide anion production using the WST-1 assay. All analyses were controlled for potential confounders. CAD patients with Type D showed higher superoxide anion production compared to CAD patients without Type D (F(1,38)=15.57, p<0.001). Complementary analyses using the Type D subscales 'negative affectivity' and 'social inhibition', and their interaction as continuous measures, showed that both Type D subscales (negative affectivity: (ß=0.48, p=0.002, R(2)=0.227); social inhibition: (ß=0.46, p=0.003, R(2)=0.208)) and their interaction (ß=0.36, p=0.022, R(2)=0.130) were associated with higher WST-1 reduction scores. Results remained significant when controlling for classical CVD risk factors (i.e. body mass index, mean arterial blood pressure), atherosclerosis severity (i.e. intima media thickness, presence of carotid plaques), and psychological factors (depressive symptom severity, chronic stress)., Conclusions: Our results indicate higher macrophage superoxide anion production in CAD patients with Type D compared to those without Type D. This may suggest a mechanism contributing to increased morbidity and mortality in CAD patients with Type D., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Innate and acquired immunity is involved in the progression of atherosclerosis. The molecular mechanisms ruling monocyte to macrophage (Mø) differentiation are not yet fully understood. Different subtypes of plaque macrophages that have differentiated from monocytes recruited from circulating blood, have been characterized based on surface epitopes. We have recently shown that LRP5, a member of the LDL receptor superfamily supporting Wnt signalling, has an important role in monocyte to macrophage differentiation. The aim of this study was to investigate whether the CD16- and CD16+ macrophage subsets found in human atherosclerotic plaques have a differential LRP5 expression/function and Wnt signalling potential. We show for the first time that LRP5 expression is significantly higher in human CD16+Mø derived from CD14(+)CD16(+) monocytes than in CD16-Mø macrophages derived from CD14(+)CD16(-) monocytes. LRP5 is not found in human healthy vessel or arterial intimal thickening but is found in advanced human atherosclerotic lesions co-localizing only with the CD16+Mø macrophage subset. LRP5 expressing macrophages infiltrate the deep layers of atherosclerotic plaques towards the intima-media boundaries showing increased migratory activity and higher phagocytic activity. The equivalent for human patrolling CD14(+)CD16(+) monocytes in mice, CD115(+)GR1(low) monocytes, also show an increased expression of LRP5. In summary, classical CD14(+)CD16(-)monocytes that differentiate into CD16-Mø do not express LRP5. Instead, human monocytes expressing LRP5 differentiate into CD16+Mø antiinflammatory macrophages. These antiinflammatory macrophages are found in advanced atherosclerotic human plaques. Thus LRP5 is a signature of the anti-inflammatory defensive phenotype of macrophages., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present article, using human monocyte-derived macrophages and cell lines containing integrated copies of the HIV-1 promoter, we show the effects of TLR3 ligands on the pro-inflammatory cytokine IL-6. We further show the effects of TLR3 ligands on HIV-1 transactivation and transcription factors involved in HIV-1 replication. This article complements the data reported by the authors, "Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways, and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication" (Bhargavan et al., 2015) [1], and the interpretation of these data can be found in the research article published by the authors in Cellular Signaling in 2015 (Bhargavan et al., 2015) [1].

Objective: Psychological states relate to changes in circulating immune cells, but associations with immune cells in peripheral tissues such as macrophages have hardly been investigated. Here, we aimed to implement and validate a method for measuring the microbicidal potential of ex vivo isolated human monocyte-derived macrophages (HMDMs) as an indicator of macrophage activation., Methods: The method was implemented and validated for two blood sampling procedures (short-term cannula insertion versus long-term catheter insertion) in 79 participants (34 women, 45 men) aged between 18 and 75 years. The method principle is based on the reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-ulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by superoxide anions, the first in a series of pathogen-killing reactive oxygen species produced by phorbol myristate acetate-activated HMDM. Cytochrome c reduction and current generation were measured as reference methods for validation purposes. We further evaluated whether depressive symptom severity (Beck Depression Inventory) and chronic stress (Chronic Stress Screening Scale) were associated with macrophage microbicidal potential., Results: The assay induced superoxide anion responses by HMDM in all participants. Assay results depended on blood sampling procedure (cannula versus catheter insertion). Interassay variability as a measure for assay reliability was 10.92% or less. WST-1 reduction scores correlated strongly with results obtained by reference methods (cytochrome c: r = 0.57, p = .026; current generation: r values ≥ 0.47, p values <.033) and with psychological factors (depressive symptom severity: r = 0.35 [cannula insertion] versus r = -0.54 [catheter insertion]; chronic stress: r = 0.36 [cannula insertion]; p values ≤ .047)., Conclusions: Our findings suggest that the implemented in vitro method investigates microbicidal potential of HMDM in a manner that is valid and sensitive to psychological measures.