Transcriptional profiling of macaque microglia reveals an evolutionary preserved gene expression program.

Microglia are tissue-resident macrophages of the central nervous system (CNS), and important for CNS development and homeostasis. In the adult CNS, microglia monitor environmental changes and react to tissue damage, cellular debris, and pathogens. Here, we present a gene expression profile of purified microglia isolated from the rhesus macaque, a non-human primate, that consists of 666 transcripts. The macaque microglia transcriptome was intersected with the transcriptional programs of microglia from mouse, zebrafish, and human CNS tissues, to determine (dis)similarities. This revealed an extensive overlap of 342 genes between the transcriptional profile of macaque and human microglia, and showed that the gene expression profile of zebrafish is most distant when compared to other species. Furthermore, an evolutionair core based on the overlapping gene expression signature from all four species was identified. This study presents a macaque microglia transcriptomics profile, and identifies a gene expression program in microglia that is preserved across species, underscoring their CNS-tailored tissue macrophage functions as innate immune cells with CNS-surveilling properties.

FAK competes for Src to promote migration against invasion in melanoma cells.

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK(Y397F) reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.

CD47 update: a multifaceted actor in the tumour microenvironment of potential therapeutic interest.

CD47 is a ubiquitous 50 kDa five-spanning membrane receptor that belongs to the immunoglobulin superfamily. This receptor, also known as integrin-associated protein, mediates cell-to-cell communication by ligation to transmembrane signal-regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also implicated in cell-extracellular matrix interactions via ligation with thrombospondins. Furthermore, CD47 is involved in many and diverse cellular processes, including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multifaceted receptor might be a central actor in the tumour microenvironment. Solid tumours are composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms. Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with CD47 on cancer cells and stromal cells.

Advanced glycation end products (AGEs) activate mast cells.

BACKGROUND AND PURPOSE: Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. EXPERIMENTAL APPROACHES: Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. KEY RESULTS: Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca(2+) increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. CONCLUSIONS AND IMPLICATIONS: Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.

Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides.

The marked improvement of several immune-mediated inflammatory diseases during pregnancy has drawn attention to pregnancy hormones as potential therapeutics for such disorders. Low molecular weight fractions derived from the pregnancy hormone human chorionic gonadotrophin (hCG) have remarkable potent immunosuppressive effects in mouse models of diabetes and septic shock. Based on these data we have designed a set of oligopeptides related to the primary structure of hCG and tested these in models of septic shock in mice and rhesus monkeys. We demonstrate that mice exposed to lipopolysaccharide (LPS) and treated subsequently with selected tri-, tetra-, penta- and hepta-meric oligopeptides (i.e. MTR, VVC, MTRV, LQGV, AQGV, VLPALP, VLPALPQ) are protected against fatal LPS-induced septic shock. Moreover, administration of a cocktail of three selected oligopeptides (LQGV, AQGV and VLPALP) improved the pathological features markedly and nearly improved haemodynamic parameters associated with intravenous Escherichia coli-induced septic shock in rhesus monkeys. These data indicate that the designed hCG-related oligopeptides may present a potential treatment for the initial hyperdynamic phase of septic shock in humans.

Amyloid beta peptides trigger CD47-dependent mast cell secretory and phagocytic responses.

Mast cells are found in the brain, where they contribute to immune responses. They have been implicated in multiple sclerosis, but their potential role in Alzheimers disease (AD), another inflammatory disease of the central nervous system, remains elusive. In the present study, we examined mast cell responses to amyloid beta (Abeta) peptides 1-40 and 1-42, the major components of the Alzheimer amyloid plaques. Rat peritoneal mast cells were used as experimental model for human brain serosal mast cells. Fibrillar Abeta1-40 and Ami1-42 peptides induced concentration-dependent exocytosis, as assessed by measurement of histamine secretion; exocytosis was reduced by pre-treatment with pertussis toxin and with antibodies against the CD47 receptor and the beta1-integrin subunit. Fibrillar Abeta1-40 and Abeta1- 42 peptides coated on heat-inactivated yeast particles and soluble fibrillar Abeta1-40 and Abeta1-42 peptides were also recognized and phagocyted by mast cells. Uptake of the peptides was decreased in the presence of 4N1, a peptide agonist of the CD47 receptor, but remained unchanged in the presence of 4NGG, a peptide derived from 4N1 which does not bind to CD47. Non-fibrillar forms of Abeta1-40 and 1-42 peptides were unable to elicit mast cell responses. These results show that fibrillar Abeta peptides can trigger mast cells and elicit exocytosis and phagocytosis. The Abeta-induced activation of mast cells operates through a CD47/beta1-integrin membrane complex coupled with Gi-protein. The present data support the hypothesis that mast cells, similarly to microglial cells, could play a major role in AD pathogenesis.

Activation of CD47 receptors causes histamine secretion from mast cells.

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.

Heptahelical and other G-protein-coupled receptors (GPCRs) signaling.

Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G-proteins) which transduce most signals through their alpha and betagamma subunits to effectors, enzymes and ion channels. Of the 367 heptahelical receptors for endogenous ligands, about 330 are potential targets for drug discovery with agonist, antagonist or inverse agonist properties. The term G-protein-coupled receptors (GPCRs) is a broader functional definition rather than a structural one referring to heptahelical receptors specifically. Non-heptahelical putative GPCRs include some transmembrane receptors with tyrosine-kinase activity on their cytosolic endings (EGF, insulin and IGF-1 receptors), other transmembrane receptors (mannose-6-phosphate/IGF-2 receptor and integrin-associated protein IAP or CD47), and some receptors belonging to the class of glycosylphosphatidylinositol (GPI)-anchored proteins and located on the outer face of the plasma membrane. Also, activators of G-protein signaling (AGS) proteins that regulate vesicular trafficking activate heterotrimeric G-proteins in the Golgi independently of receptor activation. Main effectors activated through their direct interactions with alpha subunits or betagamma dimers of heterotrimeric G-proteins include adenylylcyclases, cGMP-phosphodiesterase, phospholipases Cbeta, phosphoinositide 3-kinase gamma, Ca(V2) calcium channels, GIRK/Kir3 potassium channels, and guanine nucleotide exchange factors RasGEF and RhoGEF leading to small G-proteins and MAP-kinases activation. Current signaling cascades leading to final cell responses are depicted.

6-Methylprednisolone does not impair anti-thymocyte globulin (ATG) immunosuppressive activity in non-human primates.

BACKGROUND: Induction treatments with anti-thymocyte globulin (ATG) in solid organ transplantation may enhance the efficacy of maintenance immunosuppressive therapy. Since ATG can trigger Fas (CD95) mediated T cell apoptosis, a process antagonized in vitro by corticosteroids, an important issue is whether corticosteroids could interfere with T cell depleting and immunosuppressive activities of ATG. METHODS: MHC mismatched skin allografts were performed on cynomolgus and rhesus monkeys treated with ATG (20 mg/kg) associated or not with 6-methylprednisolone (10 mg/kg). RESULTS: There was no difference between the two immunosuppressive regimens as regards the intensity and duration of peripheral T lymphocyte depletion and the appearance of anti-ATG antibodies. Skin graft survival was increased in monkeys treated with 6-methylprednisolone as compared with ATG alone. CONCLUSIONS: In vivo, corticosteroids do not interfere with ATG ability to induce massive T cell depletion and to delay skin allograft rejection in non-human primates.

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout.

The antibacterial properties of the indigenous microflora of rainbow trout (Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum. Eight strains out of a subset of 11 antagonists caused a 3-6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13-43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.