Mechanism Of Phagocytosis Research Articles

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines.

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

Hubungan Biofilm dengan Infeksi: Implikasi pada Kesehatan Masyarakat dan Strategi Mengontrolnya

Biofilms are formed in the surfaces of mucosal of the body cavity can be a major source of infection . Infection by microbial biofilm formers are difficult to treat because of their greater resistance to antimicrobial agents than individual cells . Therefore biofilm -related infections and increased the economic burden of the country. The purpose of this literature review is to examine literature on biofilms and biofilm-associated microbes and their contribution to the disease burden of man with the aim of drawing attention to their public health implication.Biofilm are defined as “collections of microorganisms and their associated extracellular products at an interface and generally attached to a biological and non-biological substratum. Biofilm formation are influenced by factors controlling cell attachment, nature of surface, propertis of medium, and properties of the microbial cell surface. The ability of biofilm formation is genetically regulated by ica ABDC operon. Biofilm life cycle include adhesion of cells, formation of microcolonies, formation of biofilm and detachment anddispersal of biofilm. Structure of biofilm consists microorganisms and extracellular polymeric substances with vertical structures of microorganisms sometimes take the form of towers or mushrooms which are separated by interstitial spaces. Bacterial biofilm showed increased resistance to antibiotics, disinfectants and resistant to phagocytosis and other mechanisms of innate and adaptive immune system. Biofilms associated with food borne illness and affect to food security. Biofilms have been implicated in a wide variety of microbial infections in the body such as as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, legionellosis ,infections involving contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves. It is important for government agencies with a mandate for safeguarding public health and environment to develop and adopt ap propriate health risk assessment and biofilm-specific guidelines that are protective of both public health and the environment.

Phagocytic Roles of Glial Cells in Healthy and Diseased Brains.

Glial cells are receiving much attention since they have been recognized as important regulators of many aspects of brain function and disease. Recent evidence has revealed that two different glial cells, astrocytes and microglia, control synapse elimination under normal and pathological conditions via phagocytosis. Astrocytes use the MEGF10 and MERTK phagocytic pathways, and microglia use the classical complement pathway to recognize and eliminate unwanted synapses. Notably, glial phagocytosis also contributes to the clearance of disease-specific protein aggregates, such as β-amyloid, huntingtin, and α-synuclein. Here we reivew recent findings showing that glial cells are active regulators in brain functions through phagocytosis and that changes in glial phagocytosis contribute to the pathogenesis of various neurodegenerative diseases. A better understanding of the cellular and molecular mechanisms of glial phagocytosis in healthy and diseased brains will greatly improve our current approach in treating these diseases.

Overexpression of Toll-Like Receptor 4 Contributes to Phagocytosis of Salmonella Enterica Serovar Typhimurium via Phosphoinositide 3-Kinase Signaling in Sheep

Background/Aims: Phagocytosis of bacteria by monocytes/macrophages can trigger the immune response and the clearance of bacteria. This innate immune response involves Toll-like receptor 4 (TLR4). However, much remains unknown about the mechanism of TLR4-regulated phagocytosis of Salmonella enterica serovar Typhimurium (S. typhimurium) within sheep monocytes/macrophages. Here, we aimed to address these knowledge gaps by infecting transgenic sheep overexpressing TLR4 with S. typhimurium and examining the phagocytic mechanisms involved. Methods: Transgenic sheep were generated by microinjection of the constructed plasmids into fertilized eggs. Monocytes/macrophages isolated from sheep blood were stimulated with LPS and S. typhimurium. Phagocytosis-related factor expression, phagocytic ability, and adhesion were then determined. TLR4/phosphatidylinositide 3-kinase (PI3K) signaling was inhibited to investigate if this pathway is involved in changes in bacterial internalization in sheep. Results: We found that TLR4 overexpression effectively activated the PI3K signaling pathway and upregulated the expression of scavenger receptors. Additionally, actin polymerization and adhesive capacity were both enhanced in TLR4-overexpressing sheep monocytes/macrophages. TLR4 inhibition decreased S. typhimurium phagocytosis by reducing the actin polymerization and adhesive capacity of cells. Furthermore, inhibition of PI3K markedly impaired TLR4-dependent phagocytosis by restraining actin polymerization and scavenger receptor expression and reduced the adhesive capacity of the monocytes/macrophages. Conclusion: Our findings indicate that overexpression of TLR4 enhances phagocytosis through PI3K signaling and the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages infected with S. typhimurium.

Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis.

Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3β1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin β1 play a role in the activation of the Src kinase pathway. However, only integrin β1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.

What We're Reading

Exosomes (from Cancer Res 2016; 76: 1770-80).Exosomes are small extracellular vesicles that pass cellular contents from cell to cell and can contain protein-shielded RNA enriched in noncoding sequences. In the tumor microenvironment, cancer cells responsive to interferon-stimulated genes (ISGs) promoted the incorporation of unshielded RNA into exosomes released by stroma. When taken up by cancer cells, the RNA bound the pattern-recognition receptor RIG-I, activated STAT1, and induced ISGs. This unshielding and transfer illustrates a commonality between tumor– and viral–host interactions.Nabet BY, …, Minn AJ. Cell 2017 July 13; 170: 352–66.Model of C1 (from Proc Natl Acad Sci USA 2017; 114: 986–91).Therapeutic antibodies activate com plement- and Fc receptor–mediated phagocytosis and cytotoxic mechanisms, both of which could be important for their antitumor effects. A mutant form of IgG was devised whose Fc region bound complement but not Fc receptors and could kill tumor cells just as well as Abs utilizing FcRs. These antibodies avoid adverse reactions specific to FcR activation, and provide additional options for specific immunotherapies.Lee C-H, …, Georgiou G. Nat Immunol 2017 August; 18: 889–898.Staying steady - and inconspicuous (from Pixabay).Phagocytes from different developmental stages and tissues were found to run the same homeostatic program, which is initiated by IFN-γ. The coordinately regulated suite of genes was also expressed in cells found in metastatic cutaneous melanomas, with SOCS being key to limiting tumor surveillance. Thus, a native macrophage program is usurped in the tumor microenvironment and promotes immune evasion.Nirschl CJ, …, Anandasabapathy N. Cell 2017 June 29; 170: 127–41.Heterogeneous TCR profiles (from Fig. 3).Neoantigenic differences between lung tumor nodules have been associated with resistance to checkpoint blockade. Reuben et al. found, through TCR profiling of individual nodules, that most T-cell specificities were restricted to single nodules, though a few clones were shared. Internodule TCR heterogeneity correlated with risk of relapse, suggesting it has clinical significance. Manipulating T-cell access while targeting neoantigens with wide intratumoral distribution may be key to better antitumor therapies.Reuben A, …, Zhang J. Cancer Discov 2017 July 21; doi: 10.1158/2159-8290.CD-17-0256.Pre-mismatch repair (from Elizabeth Augusta via Wikimedia Commons).Eighty-six patients subjected to at least one previous therapy were enrolled in a prospective study to determine the efficacy of anti–PD-1 treatment in DNA repair–defective cancers across multiple tumor types. Overall survival after 3 years was 64% in these patients, regardless of tumor origin, supporting the use of checkpoint blockade as a standard of care for the more than 60,000 refractory patients with MMR-deficient tumors diagnosed annually in the United States.Le DT, …, Diaz LA Jr. Science 2017 June 8; doi: 10.1126/science.aan6733.Other screens (from Pixabay).CRISPR-mutated melanoma cells were screened in vivo in mice treated with GVAX + anti–PD-1 therapy, revealing proteins that increased either resistance or sensitivity to immunotherapy. IFNγ pathway deletions predictably enabled immunotherapy resistance. In contrast, loss of the phosphatase PTPN2 increased antigen presentation, recognition, and activation of CD8+ T cells. This synergy with immunotherapy suggests genetic screens can identify new immunotherapy targets.Manguso RT, …, Haining WN. Nature 2017 July 19; doi: 10.1038/nature23270.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

PHAGOCYTIC ACTIVITY AND BLOOD NEUTROPHILS RESPIRATORY BURST STATE FEATURES AMONGST WIDESPREAD PURULENT PERITONITIS PATIENTS IN THE POSTOPERATIVE PERIOD DYNAMICS

The research aim was to study the phagocytic activity and the blood neutrophils respiratory burst state amongst the patients with widespread purulent peritonitis (WPP) in the dynamics of the postoperative period. The study was involved patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP. Blood sampling was performed prior to surgery (pre-operative period) and at 7, 14 and 24 day post-operative period. The neutrophil phagocytosis level was determined by flow cytometry using FITC-labeled staphylococcal protein A. There was counted the percentage of fluorescent neutrophils (defined as the phagocytic index — PI) and average of the cell fluorescence (phagocytic number — PN). According to the fluorescence intensity divided phagocytic neutrophils in an active (high PN) and weakly phagocytic (low PN). Condition of the respiratory burst in the neutrophils was examined using a chemiluminescent assay. It was found that patients with WPP in the preoperative period had increased phagocytic activity of the blood neutrophils. The maximum phagocytic activity observed on day 7 of the postoperative period and by 24 days the number of phagocytic cells is reduced to control level whereas phagocytic activity of the cells remains at the preoperative level. In the preoperative period in patients with WPP the amount of active phagocytic blood neutrophils 3.3 times higher than the number of weakly phagocytic cells. By the end of the observation period the number of active and weakly phagocytic neutrophils returning to normal. In the study of the state of respiratory burst in the neutrophils by the WPP patients discovered that the primary synthesis levels of reactive oxygen species (ROS) increases only with 7 days of postoperative period and the induced activity of NADPH-oxidase remains elevated to the end of the observation period. At the same time, the synthesis of secondary ROS in the blood neutrophil of the WPP patients increased already in the preoperative period and remains high until the end of the survey. Using correlation analysis it was found that if healthy people had a competitive relationship between the level of synthesis of primary ROS and the activity of phagocytosis then all detected correlations in the WPP patients characterized by high level of coordination between the respiratory burst and the phagocytic activity of the neutrophils. These relationships are identified in the preoperative period, postoperatively do not exist. It can be assumed that the surgery and the subsequent treatment leads to complete incoordination in the neutrophils the mechanisms of phagocytosis and respiratory burst.

Geometry of carbon nanotubes and mechanisms of phagocytosis and toxic effects

A review of in vivo and in vitro toxicological studies of the potential toxic effects of carbon nanotubes is presented along with the analysis of experimental data and a hypothesis about the nanotube-asbestos similarity. Developments of the structure-activity paradigm have been reviewed along with the size effects and the classification of carbon nanotubes into eleven distinct classes (e.g., the high aspect ratio nanotubes, thick multi-wall nanotubes and short nanotubes). Scaling analysis of similarities between different classes of carbon nanotubes and asbestos fibers in the context of their potential toxicity and the efficiency of phagocytosis has been reviewed. The potential toxic effects of carbon nanotubes have been characterized by their normalized length, their aspect ratio and other parameters related to their inhalability, engulfment by macrophages and the effectiveness of phagocytosis. Geometric scaling parameters and the classification of carbon nanotubes are used to develop an updated parametric map for the extrapolation of the potential toxic effects resulting from the inhalation of long and short carbon nanotubes. An updated parametric map has been applied to the evaluation of the efficiency of phagocytosis involving distinct classes of carbon nanotubes. A critical value of an important nondimensional parameter characterizing the efficiency of phagocytosis for different nanotubes is presented along with its macrophage-based normalization. The present evaluation of the potential toxicological effects of the high aspect ratio carbon nanotubes is found to be in the agreement with other available studies and earlier scaling analyses.

Abstract WMP105: Human Monocyte Derived Macrophage Phagocytize Eryptotic Erythrocytes in a Phosphotidylserine Dependent Mechanism

Introduction: After intracerebral hemorrhage (ICH), erythrocytes contribute to secondary injury by releasing toxic hemoproteins. Our lab has previously shown that blood derived macrophages play an important role in ICH clearance but mechanisms of phagocytosis by human macrophages are unknown. This study aims to quantify eryptotic (phosphatidylserine (PtdSer)-expressing) red blood cells (RBCs) in an in vivo model of ICH, and to investigate the mechanisms that play a role in autologous eryptotic phagocytosis by human monocyte derived macrophages (huMDMs). Methods: ICH was induced in mice by autologous blood injection. The mice were sacrificed at 1 day after ICH. The brains were separated into hemispheres and digested into a single cell suspension for analysis by flow cytometry. Cells were stained with antibodies to cell surface markers and annexin V to quantify externalized PtdSer expression. Human monocytes were cultured with M-CSF for 7 days to generate huMDMs. Autologous RBCs were heat shocked (HS) to induce eryptosis. The huMDMs were cocultured with HS RBCs, HS RBCs treated with annexin V, or control RBCs. After 1 hour of coculture, the huMDMs were washed, stained and erythrophagocytosis quantified by microscopy. Results: The proportion of cells that externalized PtdSer increased by almost 20 fold at day 1 after ICH. Control brains mixed with fresh RBCs and subjected to tissue prep did not show PtdSer expression, ensuring that the PtdSer expression detected was induced in vivo (Fig A). HS RBCs increased PtdSer expression and were efficiently phagocytosed by huMDMs. Treatment of HS RBCs with annexin V to antagonize PtdSer-receptor interactions decreased RBC phagocytosis to levels comparable to control RBCs (Figs B and C). Conclusions: In vivo after ICH, erythrocytes externalize PtdSer, a cue to be engulfed by macrophages. Human macrophages phagocytose RBCs in a PtdSer-dependent mechanism. These findings highlight potential targets to enhance ICH clearance.

Cytological and oxidant characteristics of local cellular immunity in acute severe exacerbation of chronic obstructive pulmonary disease

The purpose of this study was to investigate cytologic and chemiluminescence (ChL) parameters of phagocytes in induced sputum and bronchoalveolar lavage fluid (BALF) of patients with severe acute exacerbation of COPD (AECOPD). Methods: Eighty-eight patients aged 45 to 75 years (56 males and 32 females) with severe AECOPD were included in the study. The control group included 16 healthy individuals. Cytological examination of induced sputum was done. The luminol-dependent ChL of BALF phagocytes obtained during bronchoscopy was investigated. Results : Leukocyte cell count and neutrophil/eosinophil ratio were increased and macrophages/lymphocyte ratio was decreased in induced sputum (p < 0.05). Exacerbations of infectious etiology were characterized by higher eosinophil count. Leukocyte cell count has been still significantly increased after resolution of the exacerbation. Eosinophil/macrophage ratio was significantly higher in bronchitis phenotype and neutrophil/lymphocyte ratio was higher in emphysema phenotype (p < 0.05). Phagocyte ChL in patients with AECOPD of infectious etiology was significantly higher compared to that in patients with AECOPD of other etiologies, both in exacerbation (spontaneous ChL, 10,049 ± 1,828 vs 8,886 ± 2,672; induced ChL, 19,912 ± 4,037 vs 10,751 ± 1,354, respectively) and in stable state (spontaneous ChL, 3,878 ± 1,147 vs 2,335 ± 1,277; induced ChL, 4,804 ± 1,284 vs 4,253 ± 1,174, respectively). This could be due to a relatively high eosinophil count in AECOPD of non-infectious etiology and in the bronchitis phenotype of COPD; eosinophils could contribute to phagocytosis and oxidative mechanisms. Conclusion . Cytological and oxidant characteristics of the airway inflammation and phagocyte-related features of the local immunity are thought to be different in AECOPD of infectious compared to non-infectious etiology and in exacerbation compared to the stable state. The luminol-dependent ChL could reliably assess phagocyte functional activity in the airway material. This method could be proposed to distinguish between infectious vs non-infectious etiologies of AECOPD in patients with bronchitis phenotype or emphysema phenotype and to evaluate the efficacy of anti-inflammatory treatment.

Phagocytosis: A Fundamental Process in Immunity.

One hundred years have passed since the death of Élie Metchnikoff (1845–1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.

FEATURES OF FREE RADICAL OXIDATION AND ANTIOXIDANT PROTECTION IN CHILDREN WITH SUGAR DIABETES OF THE FIRST TYPE

Aim. Assessment of the state of lipid peroxidation, antioxidant defense and oxidative stress indicators on the model of functional activity of neutrophil granulocytes of blood in children with type 1 diabetes mellitus depending on the stage of endocrinopathy compensation. Materials and methods. The material of laboratory-diagnostic and clinical studies was the results of a survey of 38 children without endocrine pathology and 89 children with type 1 diabetes mellitus at various stages of disease compensation. The state of antioxidant protection was assessed taking into account the overall antioxidant activity, superoxide dismutase activity, retinol level, α-tocopherol, the content of reduced and oxidized glutathione. The intensity of lipoperoxidation processes was studied by the level of substrates with conjugated double bonds, diene conjugates, ketodienes, conjugated trienes, malonic dialdehyde, common lipids. Functional activity of neutrophil granulocytes was investigated by means of luminol-dependent chemiluminescence. Results. In children with type 1 diabetes mellitus, the intensification of lipid peroxidation processes and the activation of the antioxidant defense system, including variously directed changes in non-enzymatic mechanisms, are noted against the negative effect of active oxygen species. In children with decompensated type 1 diabetes mellitus, severe lipid metabolism disorders combined with oxidative stress indices increase the course of endocrine pathology, significantly increasing the likelihood of intravascular complications. Conclusion. The coordinated generation of active forms of oxygen and the processes of oxygen-dependent metabolism of blood cells in children with compensated type 1 diabetes mellitus indicates the development of the second stage (resistance) of oxidative stress. Reduction of reactive oxygen species production, reduced activation rate оxygendependent metabolism phagocytes incompleteness phagocytosis mechanisms, correlating with an increase in lesion area (destruction of insulin-producing β-cells) of the pancreas in children with decompensated type 1 diabetes, indicating the occurrence of the third step (debilitation) oxidative stress. Violations of metabolic pathways in children with type 1 diabetes are determined by the intensity of «respiratory burst» of neutrophil granulocytes in the «Lipid peroxidation – antioxidant protection».

Immunoglobulins and their receptors, and subversion of their protective roles by bacterial pathogens.

Immunoglobulins (Igs) play critical roles in immune defence against infectious disease. They elicit potent elimination processes such as triggering complement activation and engaging specific Fc receptors present on immune cells, resulting in phagocytosis and other killing mechanisms. Many important pathogens have evolved mechanisms to subvert or evade Ig-mediated defence. One such mechanism used by several pathogenic bacteria features proteins that bind the Ig Fc region and compromise engagement of host effector molecules. Examples include different IgA-binding proteins produced by Staphylococcus aureus, Streptococcus pyogenes, and group B streptococci, all of which interact with the same interdomain region on IgA Fc. Since this region also forms the interaction site for the major human IgA-specific Fc receptor CD89, the bacteria are able to evade CD89-mediated clearance mechanisms. Similar disruption of Ig effector function by pathogen Ig-binding proteins is evident in other species. Remarkably, all the Ig-binding proteins studied in detail to date are seen to target the CH2-CH3 domain interface in the Ig Fc region, suggesting a common mode of immune evasion. A second Ig subversion mechanism that has evolved independently in numerous pathogens involves proteases that cleave Ig molecules within their hinge regions, uncoupling the antigen recognition capability of the Fab region from clearance mechanisms elicited by the Fc region. The emerging understanding of the structural basis for the recognition of Igs as substrates for these proteases and as interaction partners for Ig-binding proteins may open up new avenues for treatment or vaccination.

The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging.

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. Here, we report that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, our data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis.

Differential Gene Expression Associated with Meningeal Injury in Acute Mild Traumatic Brain Injury.

Injury to the meninges is not uncommon after traumatic brain injury (TBI), yet minimal research has been directed toward understanding the relevant biology. After a concussive event, the meninges are observed to abnormally enhance on post-contrast magnetic resonance imaging (MRI) in some patients, but not all. The aim of this work is to identify genes differentially expressed in patients with meningeal injury. Patients presenting to the emergency room with suspected TBI received a standard research MRI and blood draw within 48 h of injury. Two groups of patients were included: those with and without abnormal enhancement of the meninges on post-contrast MRI, both without other imaging findings. Groups were compared on microarray gene expression in peripheral blood samples using Affymetrix (Santa Clara, CA) and Partek Genomics Suite (Partek, Inc., St. Louis, MO) software (false discovery rate, <0.05). Forty patients were enrolled with a time from injury to MRI/blood draw of 16.8 h (interquartile range, 7.5-24.1). We observed 76 genes to be differentially expressed in patients with meningeal injury compared to those without, such as receptor for Fc fragment of IgA, multiple C2 domains, transmembrane 2, and G-protein-coupled receptor 27, which have been previously associated with initiating inflammatory mediators, phagocytosis, and other regulatory mechanisms. Post-contrast MRI is able to detect meningeal injury and has a unique biological signature observed through gene expression. These findings suggest that an acute inflammatory response occurs in response to injury to the meninges following a concussion.

CpG-ODN promotes phagocytosis and autophagy through JNK/P38 signal pathway in Staphylococcus aureus-stimulated macrophage

AimsPhagocytic and autophagic responses are critical for effective host defense against bacterial infection. Bacterial DNA which contains unmethylated Cytosine-phosphate-Guanine (CpG) motifs can trigger a variety of defense mechanisms via Toll-like receptor 9 (TLR9). Here, we aimed to investigate the underlying mechanism of TLR9-mediated phagocytosis and autophagy in Staphylococcus aureus (S.aureus)-stimulated macrophages. Main methodsThe macrophage cell line RAW264.7 or primary peritoneal macrophage was pretreated with CpG-ODN and then stimulated by S. aureus, where some of them were pretreated with SP600125 or SB203580 simultaneously. The protein expressions of TLR9, MyD88, SR-A, CD36, LC3, Beclin-1, and phosphorylated level of c-Jun N-terminal kinase (JNK), P38 and extracellular-regulated protein kinase (ERK) were detected by western blotting. The phagocytosis and LC3 punctate-structures of macrophage were observed by confocal laser scanning microscope. Key findingsCpG-ODN significantly amplified S. aureus-induced phagocytosis and autophagy of RAW264.7 and TLR9+/+ primary peritoneal macrophage as compared to that of Non-CpG treated cells, while such effect was abolished in TLR9−/− primary peritoneal macrophages. Meanwhile, CpG-ODN significantly enhanced S. aureus-induced phosphorylation of JNK and P38 but not ERK in RAW264.7. Specific inhibition of JNK or P38 by SP600125 or SB203580, dramatically down-regulated CpG-induced phagocytosis and autophagy in S. aureus-stimulated RAW264.7 and TLR9+/+ primary peritoneal macrophage, while they showed no further down-regulation of phagocytosis and autophagy in TLR9−/− primary peritoneal macrophages. SignificanceOur data indicated that CpG-ODN activates TLR9-JNK/P38 signaling to promote phagocytosis and autophagy in S. aureus-stimulated macrophages, these findings provide novel insights into how innate immune cells defend bacterial infection via TLR9.

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.

COXIELLA BURNETII PATHOGENICITY MOLECULAR BASIS

Coxiella burnetii is an obligate intracellular gram-negative bacterial pathogen, an ethiological agent of Q-fever, a zoonotic disease, elapsing as an acute (mostly atypical pneumonia) or a chronic (mostly endocarditis) form. The host range is represented by wide range of mammal, avian and arthropod species, but the main source of human infection are farm animals. The main route of infection is aerosolic. In case of contact with organism pathogen binds with phagocytal monocytic-macrophagal cell line. C. burnetii promotes maturation of specific phagolysosome-like compartment in host cell, called coxiella-containing vacuole, within this vacuole pathogen becames metabolically activated and actively replicates. Coxiella persists as metabolically inactive spore-like form in environment. Internalisation of C. burnetii occurs using actin-mediated phagocytosis and zipper mechanism. After internalization of bacteria maturation of phagolysosome-like compartment and large coxiella-containing vacuole formation occure, and vacuole can occupy nearly the whole cytoplasm of the host cell. Survivance of infected cells is important for chronic infection with C. burnetii. C. burnetii elongate the viability of host cell by two ways: it actively inhibits apoptotic signal cascades and induce pro-survival factors. Exceptthat C. burnetii involves autophagic pathway during coxiella-containing vacuole formation, and induction of autophagy promotes pathogen replication. During infection C. burnetii translocates effector substrates from bacterial cytosole to euca ryotic host cell cytosole using type IV secretion system, where effectors modulate host cell proteins. Overall approximately 130 secreted effectors of type IV transport system, but function of most of them remains unknown to date. Specific sec reted proteins for variety of strains and isolates were identified, confirmed that certain pathotypes of C. burnetii can exist. Identification and characterization of novel virulence factors it is now possible through axenic media for C. burnetii cultivation and development of site-specific mutagenesis and other genetic technics, which is important for research of C. burnetii molecular pathogenesis.

Efferocytosis of apoptotic human papillomavirus-positive cervical cancer cells by human primary fibroblasts.

Efficient clearance of apoptotic cells, named efferocytosis, is a fundamental physiological process for tissue development and homeostasis. The contribution of non-professional phagocytes like fibroblasts to efferocytosis has been established, although the underlying mechanisms are not well understood. We recently demonstrated that horizontal DNA transfer can occur through the uptake of apoptotic human papillomavirus-positive cancer cells by human primary fibroblasts leading to their transformation. The aim of this present study was to analyse the cellular and molecular mechanisms that drive the phagocytic activity of human primary fibroblasts in the context of apoptotic cervical cancer cell removal. Here we provide evidence that human primary fibroblasts engulf late more efficiently than early apoptotic cells, but their phagocytic ability remains limited compared to professional phagocytes such as human monocyte-derived macrophages. The engulfment occurs in a time-, temperature- and calcium-dependent manner. Remodelling of actin-fibers contributes to the biogenesis of apoptotic cell containing macroendocytic vacuoles. Both morphological analyses and pharmacological approaches confirmed the involvement of actin-driven phagocytosis and likely macropinocytotic mechanisms in apoptotic target internalization. The uptake of apoptotic cells requires phosphatidylserine recognition, which is mainly mediated by phosphatidylserine-receptor brain-specific angiogenesis inhibitor 1. Confocal microscopy analyses with organelle-specific markers revealed that internalised apoptotic material traffics into late phagolysosomes and specific features of microtubule-associated protein 1 light chain 3-associated phagocytosis were observed. Our in vitro data show that fibroblasts contribute to apoptotic tumour cell removal by phagocytosis and likely macropinocytotic mechanisms. Efferocytosis by fibroblasts involves phosphatidylserine receptor brain-specific angiogenesis inhibitor 1, which participates in subsequent uptake orchestration via actin cytoskeleton remodelling. Our results highlight the cellular and molecular mechanisms of fibroblast-mediated clearance of apoptotic tumour cells. Consequences regarding alternative mechanism of carcinogenesis or tumour progression should be addressed.