Lisa Rizzetto

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL‐1β in macrophages. Inflammasome activation in macrophages results from differences in cell‐wall architecture between yeasts and hyphae and is partly mediated by the dectin‐1/Syk pathway. These results define the dectin‐1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.

Inhibition of Immune Synapse by Altered Dendritic Cell Actin Distribution: A New Pathway of Mesenchymal Stem Cell Immune Regulation

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC–DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.

Adhesion to carbon nanotube conductive scaffolds forces action-potential appearance in immature rat spinal neurons.

In the last decade, carbon nanotube growth substrates have been used to investigate neurons and neuronal networks formation in vitro when guided by artificial nano-scaled cues. Besides, nanotube-based interfaces are being developed, such as prosthesis for monitoring brain activity. We recently described how carbon nanotube substrates alter the electrophysiological and synaptic responses of hippocampal neurons in culture. This observation highlighted the exceptional ability of this material in interfering with nerve tissue growth. Here we test the hypothesis that carbon nanotube scaffolds promote the development of immature neurons isolated from the neonatal rat spinal cord, and maintained in vitro. To address this issue we performed electrophysiological studies associated to gene expression analysis. Our results indicate that spinal neurons plated on electro-conductive carbon nanotubes show a facilitated development. Spinal neurons anticipate the expression of functional markers of maturation, such as the generation of voltage dependent currents or action potentials. These changes are accompanied by a selective modulation of gene expression, involving neuronal and non-neuronal components. Our microarray experiments suggest that carbon nanotube platforms trigger reparative activities involving microglia, in the absence of reactive gliosis. Hence, future tissue scaffolds blended with conductive nanotubes may be exploited to promote cell differentiation and reparative pathways in neural regeneration strategies.

Differential IL-17 Production and Mannan Recognition Contribute to Fungal Pathogenicity and Commensalism

In this study, we present evidence of differential Th17 responses in human monocyte-derived dendritic cells exposed to the pathogenic Candida albicans or the nonpathogenic Saccharomyces cerevisiae. We use different forms of the microorganisms, cells, hyphae, and spores, as a toolbox to dissect the role of surface mannan in the fungal immune response. In contrast to the S. cerevisiae yeast cell-induced Th1 response, dendritic cells stimulated with spores or C. albicans hyphae induce cellular responses shifted toward Th17 differentiation. The differential recognition of specific mannan structures is the master regulator of the discrimination between harmful and harmless fungi. The switch between spores and yeast is crucial for the commensalism of S. cerevisiae and depends on the use of a different receptor repertoire. Understanding the role of cell wall recognition during infection might lead to understanding the boundaries between safety and pathogenicity.

Strain Dependent Variation of Immune Responses to A. fumigatus: Definition of Pathogenic Species

For over a century microbiologists and immunologist have categorized microorganisms as pathogenic or non-pathogenic species or genera. This definition, clearly relevant at the strain and species level for most bacteria, where differences in virulence between strains of a particular species are well known, has never been probed at the strain level in fungal species. Here, we tested the immune reactivity and the pathogenic potential of a collection of strains from Aspergillus spp, a fungus that is generally considered pathogenic in immuno-compromised hosts. Our results show a wide strain-dependent variation of the immune response elicited indicating that different isolates possess diverse virulence and infectivity. Thus, the definition of markers of inflammation or pathogenicity cannot be generalized. The profound understanding of the molecular mechanisms subtending the different immune responses will result solely from the comparative study of strains with extremely diverse properties.

Carbon Nanotube Scaffolds Instruct Human Dendritic Cells: Modulating Immune Responses by Contacts at the Nanoscale

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

Abstract.In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed.

Using Pathway Signatures as Means of Identifying Similarities among Microarray Experiments

Widespread use of microarrays has generated large amounts of data, the interrogation of the public microarray repositories, identifying similarities between microarray experiments is now one of the major challenges. Approaches using defined group of genes, such as pathways and cellular networks (pathway analysis), have been proposed to improve the interpretation of microarray experiments. We propose a novel method to compare microarray experiments at the pathway level, this method consists of two steps: first, generate pathway signatures, a set of descriptors recapitulating the biologically meaningful pathways related to some clinical/biological variable of interest, second, use these signatures to interrogate microarray databases. We demonstrate that our approach provides more reliable results than with gene-based approaches. While gene-based approaches tend to suffer from bias generated by the analytical procedures employed, our pathway based method successfully groups together similar samples, independently of the experimental design. The results presented are potentially of great interest to improve the ability to query and compare experiments in public repositories of microarray data. As a matter of fact, this method can be used to retrieve data from public microarray databases and perform comparisons at the pathway level.

Systems biology of infectious diseases: a focus on fungal infections.

The study of infectious disease concerns the interaction between the host species and a pathogen organism. The analysis of such complex systems is improving with the evolution of high-throughput technologies and advanced computational resources. This article reviews integrative, systems-oriented approaches to understanding mechanisms underlying infection, immune response and inflammation to find biomarkers of disease and design new drugs. We focus on the systems biology process, especially the data gathering and analysis techniques rather than the experimental technologies or latest computational resources.