The possibility of using xenogeneic phagocytes in wound treatment.

Metamorphosis in the insect larva is associated with disintegration, engulf and digestion of larval tissues. These processes are accompanied by a significant shift in physiological parameters like high activity of hydrolytic enzymes and decrease of pH. In the way, the metamorphosing larva resembles the processes occurring in the wound at the stage of inflammation. Based on this thesis, we put forward the idea of the possibility of using insect phagocytes in the wound treatment. The search for a suitable insect cell line and the study of its properties were the purpose of the work. The abilities of insect phagocytes to retain viability and functional activity under conditions physiological for humans were also investigated. We found that blue blowfly Calliphora vicina larvae had histolysocytes, a specialized population of professional phagocytes involved in the histolysis. In vitro, histolysocytes possess high phagocytic activity to fragments of vertebrate soft tissues and debris. These cells retain viability and functional activity for a long time under conditions that are physiological for vertebrate cells. Moreover histolysocytes can realize the humoral control over the bacteria through the synthesis of antimicrobial peptides. So histolysocytes have the potential to be used as xenogeneic phagocytes in the wound treatment. The data obtained allow proceeding to experiments on laboratory animals for studying the effect of such therapy on the wound healing process.

A guide to measuring phagosomal dynamics.

Phagocytosis is an essential mechanism for immunity and homeostasis, performed by a subset of cells known as phagocytes. Upon target engulfment, de novo formation of specialized compartments termed phagosomes takes place. Phagosomes then undergo a series of fusion and fission events as they interact with the endolysosomal system and other organelles, in a dynamic process known as phagosome maturation. Because phagocytes play a key role in tissue patrolling and immune surveillance, phagosome maturation is associated with signaling pathways that link phagocytosis to antigen presentation and the development of adaptive immune responses. In addition, and depending on the nature of the cargo, phagosome integrity may be compromised, triggering additional cellular mechanisms including inflammation and autophagy. Upon completion of maturation, phagosomes enter a recently described phase: phagosome resolution, where catabolites from degraded cargo are metabolized, phagosomes are resorbed, and vesicles of phagosomal origin are recycled. Finally, phagocytes return to homeostasis and become ready for a new round of phagocytosis. Altogether, phagosome maturation and resolution encompass a series of dynamic events and organelle crosstalk that can be measured by biochemical, imaging, photoluminescence, cytometric, and immune-based assays that will be described in this guide.

Stearoyl-CoA desaturase-1 impairs the reparative properties of macrophages and microglia in the brain.

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination.

Ultrastructure of phagocytes and oocysts of Nematopsis sp. (Apicomplexa, Porosporidae) infecting Crassostrea rhizophorae in Northeastern Brazil.

This work describes the detailed ultrastructural morphology of the phagocyte imprisoning an oyster of Nematopsis (Apicomplexa) found in Crassostrea rhizophorae, in the city of Maceió (AL), Brazil. The highly infected hosts had half-open leaflets with weak, slow retraction of the adductor muscles. Variable number of ellipsoid oocytes, either isolated and or clustered, was found between myofibrils of the adductor muscle. Each oocyst was incarcerated in a parasitophorous vacuole of host uninucleated phagocyte. The oocysts were composed of a dense wall containing a uninucleate vermiform sporozoite. The wall of the fine oocysts was composed of homogeneous electron-lucent material formed by three layers of equal thickness, having a circular orifice-micropyle obstructed by the operculum. The oocysts presented ellipsoid morphology with their wall was surrounded by a complex network of numerous microfibrils. Important details of the taxonomic value were visualized such as the ultrastructural organization of the oocyst wall and the organization of the micropyle and operculum, beyond the microfibrils that protrude from the oocyst wall only observed by transmission electron microscopy (TEM) and that may aid in the identification of the species. However, in order to clarify the systematic position of the species reported of the genus Nematopsis, it is important to proceed with genetic analyses.

Clearance by Microglia Depends on Packaging of Phagosomes into a Unique Cellular Compartment.

Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.

Cellulose nanocrystals modulate alveolar macrophage phenotype and phagocytic function.

Nanocellulose is a promising bio-nanomaterial with attractive properties suitable for multiple industrial applications. The increased use of nanocellulose may lead to occupational exposure and negative health outcomes. However, knowledge on its health effects is limited, and while nanocellulose exposure may induce acute inflammatory responses in the lung, the underlying mechanisms are unknown. Alveolar macrophages are key cells in alveolar particle clearance. Their activation and function may be affected by various particles. Here, we investigated the uptake of pristine cellulose nanocrystals (CNC), and their effects on alveolar macrophage polarization and biological function. CNC uptake enhanced the secretion of several cytokines but did not on its own induce a complete macrophage polarization. In presence of macrophage activators, such as LPS/IFNG and IL4/IL13, CNC exposure enhanced the expression of M1 phenotype markers and the secretion of pro-inflammatory cytokines and chemokines, while decreasing M2 markers. CNC exposure also affected the function of activated alveolar macrophages resulting in a prominent cytokine burst and altered phagocytic activity. In conclusion, CNC exposure may result in dysregulation of macrophage activation and function that are critical in inflammatory responses in the lung.

Ultrastructure of phagocytes and oocysts of Nematopsis sp. (Apicomplexa, Porosporidae) infecting Crassostrea rhizophorae in Northeastern Brazil / Ultraestrutura de fagócitos e oocistos de Nematopsis sp. (Apicomplexa, Porosporidae) infectando Crassostrea rhizophorae no Nordeste brasileiro

Abstract This work describes the detailed ultrastructural morphology of the phagocyte imprisoning an oyster of Nematopsis (Apicomplexa) found in Crassostrea rhizophorae, in the city of Maceió (AL), Brazil. The highly infected hosts had half-open leaflets with weak, slow retraction of the adductor muscles. Variable number of ellipsoid oocytes, either isolated and or clustered, was found between myofibrils of the adductor muscle. Each oocyst was incarcerated in a parasitophorous vacuole of host uninucleated phagocyte. The oocysts were composed of a dense wall containing a uninucleate vermiform sporozoite. The wall of the fine oocysts was composed of homogeneous electron-lucent material formed by three layers of equal thickness, having a circular orifice-micropyle obstructed by the operculum. The oocysts presented ellipsoid morphology with their wall was surrounded by a complex network of numerous microfibrils. Important details of the taxonomic value were visualized such as the ultrastructural organization of the oocyst wall and the organization of the micropyle and operculum, beyond the microfibrils that protrude from the oocyst wall only observed by transmission electron microscopy (TEM) and that may aid in the identification of the species. However, in order to clarify the systematic position of the species reported of the genus Nematopsis, it is important to proceed with genetic analyses.

Resumo Este trabalho descreve a morfologia ultraestrutural detalhada do fagócito encarcerando um oocisto de Nematopsis (Apicomplexa) encontrado em Crassostrea rhizophorae, na cidade de Maceió (AL), Brasil. Os hospedeiros muito infectados apresentavam valvas entreabertas com retração fraca e lenta dos músculos abdutores. Número variável de oócitos de forma elipsoide, isolados e ou agrupados foi encontrado entre as miofibrilas do músculo abdutor. Cada oocisto estava encarcerado num vacúolo parasitóforo do fagócito uninucleado do hospedeiro. Os oocistos eram compostos por uma parede densa contendo um esporozoíto vermiforme uninucleado. A parede dos oocistos finos era composta de material electron-lucente homogêneo formado por três camadas de espessura igual, possuindo um orifício circular - micrópila, obstruída pelo opérculo. Os oocistos apresentavam morfologia elipsoide, sua parede era circundada por uma complexa rede de numerosas microfibrilas. Detalhes de valor taxonômico importantes foram visualizados tais como: a organização ultraestrutural da parede do oocisto e a organização da micrópila e do opérculo, além das microfibrilas que se projetam da parede do oocisto, estrutura apenas observada em microscopia eletrônica de transmissão (MET) e que pode auxiliar na identificação da espécie. Contudo, para esclarecer a posição sistemática da maioria das espécies relatadas do gênero Nematopsis é importante prosseguir com as análises genéticas.

Mononuclear phagocytes locally specify and adapt their phenotype in a multiple sclerosis model.

Mononuclear phagocytes are key regulators of both tissue damage and repair in neuroinflammatory conditions such as multiple sclerosis. To examine divergent phagocyte phenotypes in the inflamed CNS, we introduce an in vivo imaging approach that allows us to temporally and spatially resolve the evolution of phagocyte polarization in a murine model of multiple sclerosis. We show that the initial proinflammatory polarization of phagocytes is established after spinal cord entry and critically depends on the compartment they enter. Guided by signals from the CNS environment, individual phagocytes then switch their phenotype as lesions move from expansion to resolution. Our study thus provides a real-time analysis of the temporospatial determinants and regulatory principles of phagocyte specification in the inflamed CNS.

Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex.

Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the Mycobacterium tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection.

Effect of cobalt ions on the interaction between macrophages and titanium.

Inflammation and bone reduction around dental implants are described as peri-implantitis and can be caused by an inflammatory response against bacterial products and toxins. Titanium (Ti) forms aggregates with serum proteins, which activate and cause release of the cytokine interleukin (IL-1ß) from human macrophages. It was hypothesized that cobalt (Co) ions can interact in the formation of pro-inflammatory aggregates, formed by titanium. To test this hypothesis, we differentiated THP-1 cells into macrophages and exposed them to Ti ions alone or in combination with Co ions to investigate if IL-1ß release and cytotoxicity were affected. We also investigated aggregate formation, cell uptake and human biopsies with inductively coupled plasma atomic emission spectroscopy and electron microscopy. Co at a concentration of 100 µM neutralized the IL-1ß release from human macrophages and affected the aggregate formation. The aggregates formed by Ti could be detected in the cytosol of macrophages. In the presence of Co, the Ti-induced aggregates were located in the cytosol of the cultured macrophages, but outside the lysosomal structures. It is concluded that Co can neutralize the Ti-induced activation and release of active IL-1ß from human macrophages in vitro. Also, serum proteins are needed for the formation of metal-protein aggregates in cell medium. Furthermore, the structures of the aggregates as well as the localisation after cellular uptake differ if Co is present in a Ti solution. Phagocytized aggregates with a similar appearance seen in vitro with Ti present, were also visible in a sample from human peri-implant tissue. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2518-2530, 2018.

EFF-1 fusogen promotes phagosome sealing during cell process clearance in Caenorhabditis elegans.

Phagocytosis of dying cells is critical in development and immunity1-3. Although proteins for recognition and engulfment of cellular debris following cell death are known4,5, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically complex cells, such as neurons, glia and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments6,7. Thus, such cells offer unique settings to explore engulfment programme mechanisms and specificity. Here, we report that dismantling and clearance of a morphologically complex Caenorhabditis elegans epithelial cell requires separate cell soma, proximal and distal process programmes. Similar compartment-specific events govern the elimination of a C. elegans neuron. Although canonical engulfment proteins drive cell soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell-cell fusion 8 , specifically promotes distal process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically complex cells and uncover a phagosome-sealing component that promotes cell process clearance.

Topographical interrogation of the living cell surface reveals its role in rapid cell shape changes during phagocytosis and spreading.

Dramatic and rapid changes in cell shape are perhaps best exemplified by phagocytes, such as neutrophils. These cells complete the processes of spreading onto surfaces, and phagocytosis within 100 s of stimulation. Although these cell shape changes are accompanied by an apparent large increase in cell surface area, the nature of the membrane "reservoir" for the additional area is unclear. One proposal is that the wrinkled cell surface topography (which forms micro-ridges on the neutrophil surface) provides the resource for neutrophils to expand their available surface area. However, it has been problematic to test this proposal in living cells because these surface structures are sub-light microscopic. In this paper, we report the development of a novel approach, a variant of FRAP (fluorescent recovery after photo-bleaching) modified to interrogate the diffusion path-lengths of membrane associated molecules. This approach provides clear evidence that the cell surface topography changes dramatically during neutrophil shape change (both locally and globally) and can be triggered by elevating cytosolic Ca2+.

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

The cytochemical and ultrastructural characteristics of phagocytes in the Pacific oyster Crassostrea gigas.

Phagocytes have been proved to play vital roles in the innate immune response. However, the cellular characteristics of phagocytes in invertebrates, especially in molluscs, remain largely unknown. In the present study, fluorescence activated cell sorting (FACS) was employed to sort the phagocytes from the non-phagocytic haemocytes of the Pacific oyster Crassostrea gigas. The cytochemical staining analysis revealed that phagocytes were positive staining for α-naphthyl acetate esterase and myeloperoxidase, while negative staining for toluidine blue and periodic acid-Schiff. The non-phagocytic haemocytes exhibited positive staining for periodic acid-Schiff, weak positive staining for toluidine blue, but negative staining for α-naphthyl acetate esterase and myeloperoxidase. In addition, phagocytes exhibited ultrastructural cellular features similar to those of macrophages, with large cell diameter, rough cell membrane and extended pseudopodia revealed by the scanning electron microscopy, while the non-phagocytic haemocytes exhibited small cell diameter, smooth cell surface and round spherical shape. Transmission electron microscopy further demonstrated that phagocytes were abundant of cytoplasmic bodies and mitochondria, while non-phagocytic haemocytes were characterized as the comparatively large cell nucleus with contorted and condensed heterochromatin adherent to the nuclear envelope. Moreover, compared with non-phagocytic haemocytes, phagocytes exhibited significantly higher levels of intracellular cytokines, including tumor necrosis factor, interferon-like protein and interleukin-17, and significantly higher abundance of lysosome and reactive oxygen species, which were of great importance to the activation of immune response and pathogen clearance. Taken together, these findings revealed the different cytochemical and ultrastructural features between phagocytes and non-phagocytic haemocytes in C. gigas, which would provide an important clue to investigate the mechanism of phagocytosis underlying the innate immune response.

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

Inflaming the diseased brain: a role for tainted melanins.

Inflammation plays a crucial role in neurodegenerative diseases, but the irritants responsible for this response remain largely unknown. This report addressed the hypothesis that hypochlorous acid reacts with dopamine to produce melanic precipitates that promote cerebral inflammation. Spectrophotometric studies demonstrated that nM amounts of HOCl and dopamine react within seconds. A second-order rate constant for the reaction of HOCl and dopamine of 2.5 × 10(4)M(-1)s(-1) was obtained by measuring loss of dopaminergic fluorescence due to HOCl. Gravimetric measurements, electron microscopy, elemental analysis, and a novel use of flow cytometry confirmed that the major product of this reaction is a precipitate with an average diameter of 1.5 µm. Flow cytometry was also used to demonstrate the preferential reaction of HOCl with dopamine rather than albumin. Engulfment of the chlorodopamine particulates by phagocytes in vitro caused these cells to release TNFα and die. Intrastriatal administration of 10(6) particles also increased the content of TNFα in the brain and led to a 50% loss of the dopaminergic neurons in the nigra. These studies indicate that HOCl and dopamine react quickly and preferentially with each other to produce particles that promote inflammation and neuronal death in the brain.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100ß-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

Ultrastructural aspects of naturally occurring wound in the tunic of two ascidians: Ciona intestinalis and Styela plicata (Tunicata).

Efficient wound healing is essential for all animals from insects to mammals. Ciona intestinalis and Styela plicata are solitary ascidians belonging to urochordates, a subphylum that occupies a key phylogenetic position as it includes the closest relative to vertebrates. Urochordate first physical barrier against invaders is the tunic, an extracellular matrix that is constantly exposed to all kinds of insults. Thus, when damage occurs, an innate immune response is triggered to eliminate impaired tissue and potentially pathogenic microbes, and restore tissue functionality. Ultrastructural aspects of the tunic in the wound healing process of two ascidians are described. In the injured areas, we evidenced thinning of the tunic and areas of low fibre density, dense intratunic bacterial and protozoan population, and inflammatory aspects such as the increase in tunic cells, their aggregates, and phagocytosis. This is the first report on tunic physical wounding occurring in the natural habitat.

Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis.

BACKGROUND: In healthy lungs, deposited micrometer-sized particles are efficiently phagocytosed by macrophages present on airway surfaces; however, uptake of nanoparticles (NP) by macrophages appears less effective and is largely unstudied in lung disease. Using mouse models of allergic asthma and chronic obstructive pulmonary disease (COPD), we investigated NP uptake after challenge with common biogenic ambient air microparticles. METHODS: Bronchoalveolar lavage (BAL) cells from diseased mice (allergic asthma: ovalbumin [OVA] sensitized and COPD: Scnn1b-transgenic [Tg]) and their respective healthy controls were exposed ex vivo first to 3-µm fungal spores of Calvatia excipuliformis and then to 20-nm gold (Au) NP. Electron microscopic imaging was performed and NP uptake was assessed by quantitative morphometry. RESULTS: Macrophages from diseased mice were significantly larger compared to controls in OVA-allergic versus sham controls and in Scnn1b-Tg versus wild type (WT) mice. The percentage of macrophages containing AuNP tended to be lower in Scnn1b-Tg than in WT mice. In all animal groups, fungal spores were localized in macrophage phagosomes, the membrane tightly surrounding the spore, whilst AuNP were found in vesicles largely exceeding NP size, co-localized in spore phagosomes and occasionally, in the cytoplasm. AuNP in vesicles were located close to the membrane. In BAL from OVA-allergic mice, 13.9 ± 8.3% of all eosinophils contained AuNP in vesicles exceeding NP size and close to the membrane. CONCLUSIONS: Overall, AuNP uptake by BAL macrophages occurred mainly by co-uptake together with other material, including micrometer-sized ambient air particles like fungal spores. The lower percentage of NP containing macrophages in BAL from Scnn1b-Tg mice points to a change in the macrophage population from a highly to a less phagocytic phenotype. This likely contributes to inefficient macrophage clearance of NP in lung disease. Finally, the AuNP containing eosinophils in OVA-allergic mice show that other inflammatory cells present on airway surfaces may substantially contribute to NP uptake.