Internalization of titanium dioxide nanoparticles by glial cells is given at short times and is mainly mediated by actin reorganization-dependent endocytosis

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO₂ NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry.TiO₂ NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2 h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO₂ NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO₂ NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed.In conclusion, glial cells are able to internalize TiO₂ NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO₂ NPs internalization and their accumulation in brain cells could be dangerous to human health.     

DW14006 as a direct AMPKα1 activator improves pathology of AD model mice by regulating microglial phagocytosis and neuroinflammation

Alzheimer’s disease (AD) is a progressively neurodegenerative disease with typical hallmarks of amyloid β (Aβ) plaque accumulation, neurofibrillary tangle (NFT) formation and neuronal death extension. In AD brain, activated microglia phagocytose Aβ and neuronal debris, but also aggravate inflammation stress by releasing inflammatory factors and cytotoxins. Improving microglia on Aβ catabolism and neuroinflammatory intervention is thus believed to be a promising therapeutic strategy for AD. AMP-activated protein kinase (AMPK) is highly expressed in microglia with AMPKα1 being tightly implicated in neuroinflammatory events. Since indirect AMPKα1 activators may cause side effects with undesired intracellular AMP/ATP ratio, we focused on direct AMPKα1 activator study by exploring its potential function in ameliorating AD-like pathology of AD model mice. Here, we reported that direct AMPKα1 activator DW14006 (2-(3-(7-chloro-6-(2′-hydroxy-[1,1′-biphenyl]-4-yl)-2-oxo-1,2-dihydroquinolin-3-yl)phenyl)acetic acid) effectively improved learning and memory impairments of APP/PS1 mice, and the underlying mechanisms have been intensively investigated. DW14006 reduced amyloid plaque deposition by promoting microglial o-Aβ₄₂ phagocytosis and ameliorated innate immune response by polarizing microglia to an anti-inflammatory phenotype. It selectively enhanced microglial phagocytosis of o-Aβ₄₂ by upgrading scavenger receptor CD36 through AMPKα1/PPARγ/CD36 signaling and suppressed inflammation by AMPKα1/IκB/NFκB signaling. Together, our work has detailed the crosstalk between AMPKα1 and microglia in AD model mice, and highlighted the potential of DW14006 in the treatment of AD.     

Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis

ObjectiveDue to the fact that current polysaccharide-based pneumococcal vaccines have limited serotype coverage, protein-based vaccine candidates have been sought for over a decade to replace or complement current vaccines. We previously reported that a trivalent Pneumococcal Protein recombinant Vaccine (PPrV), showed protection against pneumonia and sepsis in an infant murine model. Here we investigated immunological correlates of protection of PPrV in the same model.MethodsC57BL/6J infant mice were intramuscularly vaccinated at age 1–3 weeks with 3 doses of PPrV, containing pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin mutant PlyD1. 3–4 weeks after last vaccination, serum and lung antibody levels to PPrV components were measured, and mice were intranasally challenged with a lethal dose of Streptococcus pneumoniae (Spn) serotype 6A. Lung Spn bacterial burden, number of neutrophils and alveolar macrophages, phagocytosed Spn by granulocytes, and levels of cytokines and chemokines were determined at 6, 12, 24, and 48 h after challenge.ResultsPPrV vaccination conferred 83% protection against Spn challenge. Vaccinated mice had significantly elevated serum and lung antibody levels to three PPrV components. In the first stage of pathogenesis of Spn induced pneumonia (6–24 h after challenge), vaccinated mice had lower Spn bacterial lung burdens and more phagocytosed Spn in the granulocytes. PPrV vaccination led to lower levels of pro-inflammatory cytokines IL-6, IL-1β, and TFN-α, and other cytokines and chemokines (IL-12, IL-17, IFN-γ, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden.ConclusionTrivalent PPrV vaccination results in increased serum and lung antibody levels to the vaccine components, a reduction in Spn induced lethality, enhanced early clearance of Spn in lungs due to more rapid and thorough phagocytosis of Spn by neutrophils, and correspondingly a reduction in lung inflammation and tissue damage.     

Calnexin functions in antibacterial immunity of Marsupenaeus japonicus

Calnexin (Cnx) is an endoplasmic reticulum membrane–bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca²⁺ storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity.     

人血清与中性粒细胞中乳铁蛋白含量与中性粒细胞吞噬功能的关系

用ＥＬＩＳＡ测定了人血清及中性粒细胞（ＰＭＮ）中乳铁蛋白（ＬＦ）含量，并与ＰＭＮ的吞噬功能比较，证实血清中ＬＦ水平与ＰＭＮ吞噬功能不相关，ＰＭＮ中ＬＦ含量与其吞噬、杀菌功能有密切的正相关关系。     

Human phagocytic cell response to histamine derived from potential probiotic strains of Lactobacillus reuteri

Histamine derived from lactobacilli isolates is considered to be a potential immunomodulator able to interact with the host immune system. We tested the effect of pure histamine (0.413?mM) together with the effect of cell-culture supernatants (CCS) containing different concentration of histamine produced by two of Lactobacillus reuteri isolates on the activities of antioxidant enzyme, as well as on the phagocytic activity of human leucocytes (HL). Phagocytic activity represents the non-specific immune response of HL homogenate, in vitro. Analysed histamine-producers were represented by a goatling isolate named L. reuteri KO5 and a lamb isolate named L. reuteri E and histamine production was determined using HPLC method connected with UV detection. Concretely, the samples contained the mixture of isolated HL and the addition of lactobacilli CCS at three different final concentrations of histamine ～ 0.1, 1.8 and 5.4?mM. It was found that pure histamine (0.413?mM) did not significantly influence the oxidant-antioxidant balance in HL demonstrated by unchanged degree of HL lipid peroxidation. However, at the same time, the final activity of catalase and superoxide dismutase were significantly changed (p?≤?0.001).Moreover, the phagocytic index (p?≤?0.01), lysozyme (p?≤?0.05) and peroxidase activity (p?≤?0.001), and production of IL-1β significantly decreased. CCS containing different concentration of produced histamine were also able to modulate the host non-specific immune response together with the enzymatic activity of SOD and catalase too. However, our findings indicated that the impact of lactobacilli histamine is strictly strain-dependent and concentration dependent. Moreover, it seems that histamine is not the only one lactobacilli metabolite, which may play an important role in overall immunomodulatory and antioxidant potential of tested lactobacilli.     

Comparative study of respiratory burst induced by phorbol ester and zymosan in human granulocytes

OBJECTIVES: We have comparatively evaluated respiratory burst and some steps of its signaling in human granulocytes towards different stimuli. MATERIALS AND METHODS: An alternative method to remove erythrocytes by dextran differential sedimentation was employed. Respiratory burst (RB) was assessed in a flow cytometer by the oxidation of a fluorescent probe (dichlorodihydrofluorescein). Stimuli were phorbol ester (PMA) and zymosan. RESULTS: Granulocytes obtained with dextran sedimentation mounted a normal RB for the two stimuli but cells obtained by erythrocyte lysis were ineffective to respond to zymosan (P < 0.05). EGTA did not affect PMA-induced, but inhibited zymosan-induced RB (P < 0.05). PMA-induced RB was blocked by a protein kinase C inhibitor (P < 0.05), whereas zymosan was not. Microfilament integrity was essential for zymosan but not for PMA, whereas microtubule depolymerization does not seem to be essential for both stimuli. Granulocytes obtained from recently diagnosed leukemia patients responded relatively well to both stimuli but after chemotherapy, the response to zymosan was increased whereas cells were unable to respond to PMA. CONCLUSION: The use of the proposed method allows the study of both RB and phagocytosis, making it useful to study these granulocyte responses in the clinical and experimental settings.     

The Evolving Biology of Microglia in Alzheimer’s Disease

Alzheimer’s disease (AD) is typified by a robust microglial-mediated inflammatory response within the brain. Indeed, microglial accumulation around plaques in AD is one of the classical hallmarks of the disease pathology. Although microglia have the capacity to remove β-amyloid deposits and alleviate disease pathology, they fail to do so. Instead, they become chronically activated and promote inflammation-mediated impairment of cognition and cytotoxicity. However, if microglial function could be altered to engage their phagocytic response, promote their tissue maintenance functions, and prevent release of factors that promote tissue damage, this could provide therapeutic benefit. This review is focused on the current knowledge of microglial homeostatic mechanisms in AD, and mechanisms involved in the regulation of microglial phenotype in this context.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0316-8) contains supplementary material, which is available to authorized users.     

Evidence of impaired polymorphonuclear leucocyte phagocytic functions and chemiluminescence response in rheumatoid arthritis

Summary In this study, the phagocytic uptake of ³H-thymidine-labelled Staphylococcus aureus and bacterial killing (Bk) by polymorphonuclear leucocytes (PMN) from patients with rheumatoids arthritis (RA) were investigated and compared to the luminol-enhanced chemiluminescence response (LCL) to phorbolmyristic acetate (PMA), a receptor and second message-independent activator of respiratory burst activity in definite or classical rheumatoid arthritis (RA), with regard to the inflammatory activity of the disease process, and compared to patients with osteoarthritis (OA) and healthy controls. PMN of RA patients showed a significant reduction in uptake (57.8±4.4%) and less Bk capacity (27.3±4.2%) compared to OA patients (uptake 71.4±4.3%, Bk 20.6±2.9%; P<0.001) and controls (uptake 73.2±5.2%, Bk19.3±4.2%;P<0.001). In contrast, PMN LCL response was markedly enhanced in RA patients compared to OA patients (P<0.001) and controls (P<0.001). There was no significant influence of inflammatory activity on various PMN functions in RA and no difference was found between OA and control subjects. These data clearly demonstrated impaired PMN phagocytic functions (uptake, Bk) and enhanced LCL in RA, suggesting priming and/or activated peripheral blood PMN, which might be of clinical importance concerning altered host defence in these patients.