Microglial responses after phagocytosis: Escherichia coli bioparticles,but not cell debris or amyloid beta,induce matrix metalloproteinase-9 secretion in cultured rat primary microglial cells

Upon infection or brain damage, microglia are activated to play roles in immune responses, including phagocytosis and soluble factor release. However, little is known whether the event of phagocytosis could be a trigger for releasing soluble factors from microglia. In this study, we tested if microglia secrete a neurovascular mediator matrix metalloproteinase-9 (MMP-9) after phagocytosis in vitro. Primary microglial cultures were prepared from neonatal rat brains. Cultured microglia phagocytosed Escherichia coli bioparticles within 2 hr after incubation and started to secrete MMP-9 at around 12 hr after the phagocytosis. A TLR4 inhibitor TAK242 suppressed the E. coli-bioparticle-induced MMP-9 secretion. However, TAK242 did not change the engulfment of E. coli bioparticles in microglial cultures. Because lipopolysaccharides (LPS), the major component of the outer membrane of E. coli, also induced MMP-9 secretion in a dose–response manner and because the response was inhibited by TAK242 treatment, we assumed that the LPS-TLR4 pathway, which was activated by adhering to the substance, but not through the engulfing process of phagocytosis, would play a role in releasing MMP-9 from microglia after E. coli bioparticle treatment. To support the finding that the engulfing step would not be a critical trigger for MMP-9 secretion after the event of phagocytosis in microglia, we confirmed that cell debris and amyloid beta were both captured into microglia via phagocytosis, but neither of them induced MMP-9 secretion from microglia. Taken together, these data demonstrate that microglial response in MMP-9 secretion after phagocytosis differs depending on the types of particles/substances that microglia encountered.     

Carbon monoxide controls microglial erythrophagocytosis by regulating CD36 surface expression to reduce the severity of hemorrhagic injury

Microglial erythrophagocytosis is crucial in injury response to hemorrhagic stroke. We hypothesized that regulation of microglial erythrophagocytosis via HO-1/CO depends on a pathway involving reactive oxygen species (ROS) and CD36 surface-expression. The microglial BV-2 cell line and primary microglia (PMG) were incubated +/−blood and +/−CO-exposure. PMG isolated from tissue-specific HO-1-deficient (LyzM-Cre-Hmox1 ^fl/fl) and CD36 ^−/− mice or siRNA against AMPK (AMP-activated protein kinase) were used to test our hypothesis. In a murine subarachnoid hemorrhage (SAH) model, we compared neuronal injury in wild-type and CD36 ^−/− mice. Readouts included vasospasm, microglia activation, neuronal apoptosis, and spatial memory. We observed increased microglial HO-1-expression after blood-exposure. A burst in ROS-production was seen after CO-exposure, which led to increased amounts of phosphorylated AMPK with subsequently enhanced CD36 surface-expression. Naïve PMG from LyzM-Cre-Hmox1 ^fl/fl mice showed reduced ROS-production and CD36 surface-expression and failed to respond to CO with increased CD36 surface-expression. Lack of HO-1 and CD36 resulted in reduced erythrophagocytosis that could not be rescued with CO. Erythrophagocytosis was enhanced in BV-2 cells in the presence of exogenous CO, which was abolished in cells treated with siRNA to AMPK. CD36 ^−/− mice subjected to SAH showed enhanced neuronal cell death, which resulted in impaired spatial memory function. We demonstrate that microglial phagocytic function partly depends on a pathway involving HO-1 with changes in ROS-production, phosphorylated AMPK, and surface expression of CD36. CD36 was identified as a crucial component in blood clearance after hemorrhage that ultimately determines neuronal outcome. These results demand further investigations studying the potential neuroprotective properties of CO.     

Microinjection of a growth factor cocktail affects activated microglia in the neocortex of adult rats

Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.     

Immunomodulation of RAW 264.7 Murine Macrophage Functions and Antioxidant Activities of 11 Plant Extracts

A group of 11 medicinal plants, including Lavandula pubescens, Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Silene succulenta, Silene villosa, Bogonvillea glabra, Cakile maritime, Gomphrene celesoids, Mirabilis jalaba, and Silene nocturna growing in Egypt, were extracted and examined for their immunomodulatory and antioxidant activities. RAW 264.7 cells were recruited to investigate the immunomodulatory effect through multiple parameters analysis. First, the proliferation index of macrophages cells was evaluated revealing that Trigonella foenugricium, Silene succulenta and Silene villosa have a significant cytotoxic effect on RAW cells. Interestingly, we observed enhancement of macrophages phagocytic function of by all extracts except Cakile maritime, Gomphrena celosioides and Silene nocturna. Afterwards, macrophages were challenged by incubation with LPS and the effect of various extracts on inflammatory responses was investigated; the generation of NO from activated macrophage was substantially suppressed by 7 extracts namely, Trigonella foenugricium, Calligonum comosum, Silene succulenta, Bougainvillea glabra, Mirabilis jalaba, Gomphrena celosioides and Silene nocturna. TNF-α was decreased by percentage range from 3.8 to 85.8% and Trigonella foenugricium extract showed the highest inhibition of TNF-α release. All extracts except Trigonella foenugricium, Salsola schweinforthi, Silene succulenta and Mirabilis jalaba significantly inhibited COX-2 production from stimulated macrophage. Moreover, evaluating the potential antioxidant activity of these extracts showed that Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba exhibited some antioxidant activities. Taken together, our results suggest that some of these extracts may have a considerable antinflammatory and antioxidant effects and may be a potential therapeutic choice in the treatment of inflammatory diseases.     

牛至油增强小鼠免疫功能的实验

目的：研究牛至油对小鼠免疫功能的影响。方法：采用ConA诱导的小鼠脾淋巴细胞转化实验（MTT法）、NK细胞活性测定、小鼠腹腔巨噬细胞吞噬鸡红细胞实验等方法考察牛至油对小鼠免疫功能的作用。结果：牛至油在促进NK细胞活性和单核-巨噬细胞方面的具有明显的作用，但在ConA诱导的小鼠脾淋巴细胞转化能力试验中并未表现出显著的差异。结论：牛至油确实具有增强机体免疫功能的作用。     

Cargo-laden erythrocyte ghosts target liver mediated by macrophages

Liver-targeted cargo delivery possesses great potential for the treatment of liver disease. It is urgent to find an efficient and biocompatible liver targeted delivery system. This study focused on the liver targeting properties of erythrocyte ghosts and its possible mechanism. Herein, we optimized conditions to fabricate human and mouse erythrocyte ghosts with sufficient room capable of incorporating various model substances. Erythrocyte ghosts are biocompatible cargo carriers because it is derived from autologous red blood cells (RBCs), and the cell size, zeta potential, and biconcave-disk shape of the ghosts were consistent with those of RBCs. An in vivo imaging system and positron emission tomography/computed tomography imaging showed that the ghosts were captured mainly in the liver by intravenous injection of fluorescence or ¹⁸F-fluorodeoxyglucose (FDG)-labelled ghosts into mice. In contrast, the main concentration of naked octreotide was trapped in the lungs while naked ¹⁸F-FDG was trapped in the heart. However, the concentration of cargo-loaded ghosts decreased significantly in the liver in macrophage-depleted mice. Accordingly, in vitro experiments showed that higher phosphatidylserine exposure was observed in the ghosts (38.9 %) compared to normal erythrocytes (0.69 %), and the phagocytic activity of the macrophage RAW 264.7. on the ghosts was significantly higher than that of normal erythrocytes (p < 0.001). Together they indicate that erythrocyte ghosts show liver targeting properties, and possibly owing to macrophage phagocytosis. This promising and effective therapeutic delivery system may provide therapeutic benefits for liver disease.     

Central nervous system diseases related to pathological microglial phagocytosis

Microglia are important phagocytes of the central nervous system (CNS). They play an important role in protecting the CNS by clearing necrotic tissue and apoptotic cells in many CNS diseases. However, recent studies have found that microglia can phagocytose parts of neurons excessively, such as the neuronal cell body, synapse, or myelin sheaths, before or after the onset of CNS diseases, leading to aggravated injury and impaired tissue repair. Meanwhile, reduced phagocytosis of synapses and myelin results in abnormal circuit connections and inhibition of remyelination, respectively. Previous studies focused primarily on the positive effects of microglia phagocytosis, whereas only a few studies have focused on the negative effects. In this review, we use the term "pathological microglial phagocytosis" to refer to excessive or reduced phagocytosis by microglia that leads to structural or functional abnormalities in target cells and brain tissue. The classification of pathological microglial phagocytosis, the composition, and activation of related signaling pathways, as well as the process of pathological phagocytosis in various kinds of CNS diseases, are described in this review. We hypothesize that pathological microglial phagocytosis leads to aggravation of tissue damage and negative functional outcome. For example, excessive microglial phagocytosis of synapses can be observed in Alzheimer's disease and schizophrenia, leading to significant synapse loss and memory impairment. In Parkinson's disease, ischemic stroke, and traumatic brain injury, excessive microglial phagocytosis of neuronal cell bodies causes impaired gray matter recovery and sensory dysfunction. We therefore believe that more studies should focus on the mechanism of pathological microglial phagocytosis and activation to uncover potential targets of therapeutic intervention.