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Concept: Phagocytosis

169

BACKGROUND: Antibody opsonization of Plasmodium falciparum-infected erythrocytes (IE) plays a crucial role in anti-malarial immunity by promoting clearance of blood-stage infection by monocytes and macrophages. The effects of phagocytosis of opsonized IE on macrophage proinflammatory cytokine responses are poorly understood. METHODS: Phagocytic clearance, cytokine response and intracellular signalling were measured using IFN-gamma-primed human monocyte-derived macrophages (MDM) incubated with opsonized and unopsonized trophozoite-stage CS2 IE, a chondroitin sulphate-binding malaria strain. Cytokine secretion was measured by bead array or ELISA, mRNA using quantitative PCR, and activation of NF-kappaB by Western blot and electrophoretic mobility shift assay. Data were analysed using the Mann-Whitney U test or the Wilcoxon signed rank test as appropriate. RESULTS: Unopsonized CS2 IE were not phagocytosed whereas IE opsonized with pooled patient immune serum (PPS) were (Phagocytic index (PI)=18.4, [SE 0.38] n=3). Unopsonized and opsonized IE induced expression of TNF, IL-1beta and IL-6 mRNA by MDM and activated NF- kappaB to a similar extent. Unopsonized IE induced secretion of IL-6 (median= 622 pg/ml [IQR=1,250-240], n=9) but no IL-1beta or TNF, whereas PPS-opsonized IE induced secretion of IL-1beta (18.6 pg/mL [34.2-14.4]) and TNF (113 pg/ml [421-17.0]) and increased IL-6 secretion (2,195 pg/ml [4,658-1,095]). Opsonized, but not unopsonized, CS2 IE activated caspase-1 cleavage and enzymatic activity in MDM showing that Fc receptor-mediated phagocytosis activates the inflammasome. MDM attached to IgG-coated surfaces however secreted IL-1beta in response to unopsonized IE, suggesting that internalization of IE is not absolutely required to activate the inflammasome and stimulate IL-1beta secretion. CONCLUSIONS: It is concluded that IL-6 secretion from MDM in response to CS2 IE does not require phagocytosis, whereas secretion of TNF and IL-1beta is dependent on Fcgamma receptor-mediated phagocytosis; for IL-1beta, this occurs by activation of the inflammasome. The data presented in this paper show that generating antibody responses to blood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fcgamma receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response.

Concepts: Immune system, Inflammation, Antibody, Phagocytosis, Apoptosis, Mann–Whitney U, Non-parametric statistics, Opsonin

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Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.

Concepts: Immune system, White blood cell, Antibody, Vaccine, Phagocytosis, Macrophage, Apoptosis, Opsonin

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Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Concepts: Immune system, Phagocytosis, Apoptosis, Neurology, Temporal lobe, Hippocampus, Epilepsy, Temporal lobe epilepsy

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Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein. Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction. However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2-null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm(cre) on an Mecp2-null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2-expressing microglia within the context of an Mecp2-null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2(+/-) females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.

Concepts: Gene, Cancer, Phagocytosis, Autism, Asperger syndrome, Rett syndrome, Autism spectrum, Microglia

34

Foreign particles and cells are cleared from the body by phagocytes that must also recognize and avoid clearance of “self” cells. The membrane protein CD47 is reportedly a “marker of self” in mice that impedes phagocytosis of self by signaling through the phagocyte receptor CD172a. Minimal “Self” peptides were computationally designed from human CD47 and then synthesized and attached to virus-size particles for intravenous injection into mice that express a CD172a variant compatible with hCD47. Self peptides delay macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors. Self-peptide affinity for CD172a is near the optimum measured for human CD172a variants, and Self peptide also potently inhibits nanoparticle uptake mediated by the contractile cytoskeleton. The reductionist approach reveals the importance of human Self peptides and their utility in enhancing drug delivery and imaging.

Concepts: Immune system, Antibody, Protein, Pharmacology, Cell membrane, Phagocytosis, Peptide, Phagocyte

28

Many patients with non-eosinophilic asthma have increased numbers of neutrophils in the airways. The explanation for this chronic inflammation remains unclear, but may result from an impaired ability of alveolar macrophages to phagocytose apoptotic cells (a process termed ‘efferocytosis’), as we have shown in chronic obstructive pulmonary disease (COPD).

Concepts: Immune system, Monocyte, Asthma, Pneumonia, Phagocytosis, Macrophage, Apoptosis, Chronic obstructive pulmonary disease

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Rlyso, a highly selective and sensitive pH sensor, can stain lysosomes with a novel lysosome-locating group, methylcarbitol. Rlyso was successfully used to detect lysosomal pH changes during apoptosis or induced by chloroquine while avoiding the “alkalizing effect” on lysosomes of current lysosomal probes with nitrogen-containing sidechains.

Concepts: Phagocytosis, PH, Lysosome, Vesicle

28

Cryptococcus is a potentially fatal fungal pathogen and a leading cause of death in immunocompromised patients. As an opportunistic and facultative intracellular pathogen of humans, Cryptococcus exhibits a complex set of interactions with the host immune system in general and macrophages in particular. Cryptococcus is resistant to phagocytosis but is also able to survive and proliferate within the mature phagolysosome. It can cause the lysis of host cells, can be transferred between macrophages or exit non-lytically via vomocytosis. Efficient phagocytosis is reliant on opsonisation and Cryptococcus has a number of anti-phagocytic strategies including formation of titan cells and a thick polysaccharide capsule. Following uptake, phagosome maturation appears to occur normally, but the internalised pathogen is able to survive and replicate. Here we review the interactions and host manipulation processes that occur within cryptococcal-infected macrophages and highlight areas for future research.

Concepts: Immune system, Antibody, Bacteria, Cell biology, Phagocytosis, Phagosome, Phagolysosome, Macrophage

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The incidence of fungal infections due to C. parapsilosis and closely related cryptic species (-psilosis complex) has increased in the last few years, but differences in virulence among these species have not been widely studied. Fifteen clinical isolates of C. parapsilosis, C. orthopsilosis, and C. metapsilosis, including the type strains, were used to evaluate their virulence in Galleria mellonella larvae. Fluctuations in the hemocyte density and in the phagocytic activity were also tested. Differences in the median survival for these species were demonstrated at 37 °C (2.6 ± 1.02, 2.3 ± 0.92, and 4.53 ± 1.65 d for C. parapsilosis, C. orthopsilosis, and C. metapsilosis, respectively). Galleria mellonella hemocytes phagocytosed C. metapsilosis strains more effectively than did for C. orthopsilosis and C. parapsilosis (P<0.05). The phagocytosis rate was lower for C. parapsilosis than for C. orthopsilosis (P<0.05). The hemocyte density was increased in larvae infected with C. metapsilosis compared with those infected with C. parapsilosis or C. orthopsilosis (P<0.05). Moreover, in vitro studies of virulence factors such as pseudo-hyphae production and hydrolytic enzyme secretion showed that the capability of C. metapsilosis strains to produce those virulence factors was reduced. Infections due to -psilosis complex species produced tissue damage in G. mellonella and pseudo-hyphae could be also observed during infection with C. parapsilosis.

Concepts: Immune system, Enzyme, Microbiology, Infection, Phagocytosis, Transmission and infection of H5N1, In vitro, Candida parapsilosis

27

In the phagocytosis field, ROS production by the phagocyte NOX has been associated with pathogen killing for the last 50 years. Since the discovery of nonphagocyte NOX, numerous other roles for ROS production have been identified. Oxidative stress and ROS-mediated signaling have received much attention in recent years. Much lower concentrations of ROS may be required for signaling compared with microbial killing. Based on the discoveries in nonphagocytic cells, it became logical to look for ROS functions distinct from pathogen killing, even in phagocytes. ROS are now linked to various forms of cell death, to chemotaxis, and to numerous modifications of cellular processes, including the NOX itself. ROS functions are clearly concentration-dependent over a wide range of concentrations. How much is required for which function? Which species are required for how much time? Is ROS signaling only a side effect of bactericidal ROS production? One major obstacle to answer these questions is the difficulty of reliable quantitative ROS detection. Signal transduction often takes place on a subcellular scale over periods of seconds or minutes, so the detection methods need to provide appropriate time and space resolution. We present examples of local ROS production, decreased degradation, signaling events, and potentially ROS-sensitive functions. We attempt to illustrate the current limitations for quantitative spatiotemporal ROS detection and point out directions for ongoing development. Probes for localized ROS detection and for combined detection of ROS, together with protein localization or other cellular parameters, are constantly improved.

Concepts: Immune system, Antibody, Time, Protein, Bacteria, Cell biology, Phagocytosis, Phagocyte