Gene expression profiling reveals a conserved microglia signature in larval zebrafish

International audienceMicroglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macro-phages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species. K E Y W O R D S brain, evolution, microglia, RNA sequencing, transcriptome, zebrafis

Proton transfer unlocks inactivation in cyclic nucleotide-gated A1 channels

Key points: Desensitization and inactivation provide a form of short-term memory controlling the firing patterns of excitable cells and adaptation in sensory systems. Unlike many of their cousin K+ channels, cyclic nucleotide-gated (CNG) channels are thought not to desensitize or inactivate. Here we report that CNG channels do inactivate and that inactivation is controlled by extracellular protons. Titration of a glutamate residue within the selectivity filter destabilizes the pore architecture, which collapses towards a non-conductive, inactivated state in a process reminiscent of the usual C-type inactivation observed in many K+ channels. These results indicate that inactivation in CNG channels represents a regulatory mechanism that has been neglected thus far, with possible implications in several physiological processes ranging from signal transduction to growth cone navigation. Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). Unlike many of their cousin K+ channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo < 7 the I-V relationships are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underlines current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline, suggesting analogies with the C-type inactivation observed in K+ channels. Analysis with Markovian models indicates that the non-independent binding of two protons within the transmembrane electrical field explains both the voltage-dependent blockage and the inactivation. Low pH, by inhibiting the CNGA1 channels in a state-dependent manner, may represent an unrecognized endogenous signal regulating CNG physiological functions in diverse tissues

Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization

Increasing production and application of nanomaterials raises significant questions regarding the potential for cellular entry and toxicity of nanoparticles. It was observed that the presence of serum reduces the cellular association of 20 nm carboxylate-modified fluorescent polystyrene beads up to 20-fold, relative to cells incubated in serum-free media. Analysis by confocal microscopy demonstrated that the presence of serum greatly reduces the cell surface association of nanoparticles, as well as the potential for internalization. However, both in the presence and absence of serum, nanoparticle entry depends upon clathrin-mediated endocytosis. Finally, experiments performed with cells cooled to 4°C suggest that a proportion of the accumulation of nanoparticles in cells was likely due to direct permeabilization of the plasma membrane

Gene therapy for liver diseases: recent strategies for treatment of viral hepatitis and liver malignancies

Gene therapy has emerged as a powerful and very plastic tool to regulate biological functions in diseased tissues with application in virtually all medical fields. An increasing number of experimental and clinical studies underline the importance of genes as curative agents in the future. However, intense research is needed to evaluate the potential of gene therapy to improve efficacy and minimise the toxicity of the procedure

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells

OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-gamma levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies

Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12

Despite the efficacy of IL-12 in cancer experimental models, clinical trials with systemic recombinant IL-12 showed unacceptable toxicity related to endogenous IFNgamma production. We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in a number of mouse pure strains over a wide range of doses. For instance at 2.5 x 10(9) p.f.u., systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. Much higher IFNgamma concentrations in serum samples of C57BL/6 than in those from identically treated BALB/c were found. Causes for heterogeneous toxicity can be traced to differences among murine strains in the levels of gene transduction achieved in the liver, as assessed with adenovirus coding for reporter genes. In accordance, IL-12 serum concentrations are higher in susceptible mice. In addition, sera from C57BL/6 mice treated with AdCMVmIL-12 showed higher levels of IL-18, a well-known IFNgamma inducer. Interestingly, lethal toxicity in C57BL/6 mice was abolished by administration of blocking anti-IFNgamma mAbs and also by simultaneous depletion of T cells, NK cells, and macrophages. These observations together with the great dispersion of IFNgamma produced by human PBMCs upon in vitro stimulation with IL-12, or infection with recombinant adenovirus encoding IL-12, suggest that patients might also show heterogeneous degrees of toxicity in response to IL-12 gene transfer

Tuning electrical properties of hierarchically assembled Al-doped ZnO nanoforests by room temperature Pulsed Laser Deposition

Large surface area, 3D structured transparent electrodes with effective light management capability may represent a key component in the development of new generation optoelectronic and energy harvesting devices. We present an approach to obtain forest-like nanoporous/hierarchical Al-doped ZnO conducting layers with tunable transparency and light scattering properties, by means of room temperature Pulsed Laser Deposition in a mixed Ar:O2 atmosphere. The composition of the background atmosphere during deposition can be varied to modify stoichiometry-related defects, and therefore achieve control of electrical and optical properties, while the total background pressure controls the material morphology at the nano- and mesoscale and thus the light scattering properties. This approach allows to tune electrical resistivity over a very wide range (10^-1 - 10^6 Ohm*cm), both in the in-plane and cross-plane directions. Optical transparency and haze can also be tuned by varying the stoichiometry and thickness of the nano-forests