Alessandro Michelucci

Characterization of the microglial phenotype under specific pro-inflammatory and anti-inflammatory conditions: Effects of oligomeric and fibrillar amyloid-β

M1 and M2 are the extremes of the differentiation spectrum of activated macrophages. Since microglia are members of the same cell lineage, we have characterized their transcription profile and their phagocytic activity under different conditions. LPS or IFN-gamma induce a M1-like phenotype, while IL-10 or IL-4 differentiate microglia towards a M2-deactivated or M2-alternatively-activated phenotype respectively. These differentiation processes also affect the Notch pathway. In order to study the polarization induced by Abeta, microglia was stimulated with different forms of the peptide. The oligomeric Abeta is a stronger M1-inductor than the fibrillar form. Moreover, a cytokine-induced anti-inflammatory environment reduces the microglial reactivity towards oligomeric Abeta.

Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

Notch signaling modulates the activation of microglial cells

The Notch signaling pathway plays a crucial role in specifying cellular fate in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in hematopoietic cell development. Here, we report that the Notch pathway is expressed and active in microglial cells. During inflammatory activation, the transcription of the Notch down‐stream effector Hes1 is downregulated. When Notch1 transcription in microglia is inhibited, an upregulation of the expression of pro‐inflammatory cytokines is observed. Notch stimulation in activated microglia, using a soluble form of its ligand Jagged1, induces a decrease in pro‐inflammatory cytokines secretion and nitric oxide production as well as an increase in phagocytic activity. Notch‐stimulation is accompanied by an increase in the rate of STAT3 phosphorylation and nuclear translocation. Our results show that the Notch pathway plays an important role in the control of inflammatory reactions in the CNS.

Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels

Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1−/− mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.

Jagged1 regulates the activation of astrocytes via modulation of NFκB and JAK/STAT/SOCS pathways

The Notch pathway is implicated in many aspects of the central nervous system (CNS) development and functions. Recently, we and others identified the Notch pathway to be involved in inflammatory events of the CNS. To understand the implication of this pathway on astrocytes, we have studied the Jagged‐Notch‐Hes pathway under inflammatory conditions. LPS exposure induced an upregulation of Jagged1 expression on cultured astrocytes. To address the role of Jagged1 in the modulation of inflammation, we used a siRNA mediated silencing of Jagged1 (siRNA J1). Jagged1 inhibition induced important variations on the Notch pathway components like Hes1, Hes5, Notch3, and RBP‐Jκ. siRNA J1 repressed the mRNA expression of genes known as hallmarks of the gliosis like GFAP and endothelin(B) receptor. On activated astrocytes, the inhibition of Jagged1 had antiinflammatory effects and resulted in a decrease of LPS‐induced proinflammatory cytokines (IL1β, IL1α, and TNFα) as well as the iNOS expression. The inhibition of Jagged1 induced a modulation of the JAK/STAT/SOCS signaling pathway. Most interestingly, the siRNA J1 decreased the LPS‐induced translocation of NFκB p65 and this could be correlated to the phosphorylation of IκBα. These results suggest that during inflammatory and gliotic events of the CNS, Jagged1/Notch signaling sustains the inflammation mainly through NFκB and in part through JAK/STAT/SOCS signaling pathways.

Itaconic Acid: The Surprising Role of an Industrial Compound as a Mammalian Antimicrobial Metabolite

Itaconic acid is well known as a precursor for polymer synthesis and has been involved in industrial processes for decades. In a recent surprising discovery, itaconic acid was found to play a role as an immune-supportive metabolite in mammalian immune cells, where it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. Although the immune-responsive gene 1 protein (IRG1) has been associated to immune response without a mechanistic function, the critical link to itaconic acid production through an enzymatic function of this protein was only recently revealed. In this review, we highlight the history of itaconic acid as an industrial and antimicrobial compound, starting with its biotechnological synthesis and ending with its antimicrobial function in mammalian immune cells.

Microglial activation depends on beta‐amyloid conformation: role of the formylpeptide receptor 2

J. Neurochem. (2010) 114, 576–586.

Cellular and Molecular Characterization of Microglia: A Unique Immune Cell Population

Microglia are essential for the development and function of the adult brain. Microglia arise from erythro-myeloid precursors in the yolk sac and populate the brain rudiment early during development. Unlike monocytes that are constantly renewed from bone marrow hematopoietic stem cells throughout life, resident microglia in the healthy brain persist during adulthood via constant self-renewal. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system, make microglia a unique cell population. Supporting this notion, recent genome-wide transcriptional studies revealed specific gene expression profiles clearly distinct from other brain and peripheral immune cells. Here, we highlight the breakthrough studies that, over the last decades, helped elucidate microglial cell identity, ontogeny, and function. We describe the main techniques that have been used for this task and outline the crucial milestones that have been achieved to reach our actual knowledge of microglia. Furthermore, we give an overview of the “microgliome” that is currently emerging thanks to the constant progress in the modern profiling techniques.

Gene Regulatory Network Inference of Immunoresponsive Gene 1 (IRG1) Identifies Interferon Regulatory Factor 1 (IRF1) as Its Transcriptional Regulator in Mammalian Macrophages

Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. Its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (IRG1/CAD), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (TCA) cycle intermediate. Itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the TCA cycle and enables bacteria to survive on limited carbon conditions. To elucidate the mechanisms underlying itaconic acid production through IRG1 induction in macrophages, we examined the transcriptional regulation of IRG1. To this end, we studied IRG1 expression in human immune cells under different inflammatory stimuli, such as TNFα and IFNγ, in addition to lipopolysaccharides. Under these conditions, as previously shown in mouse macrophages, IRG1/CAD accumulates in mitochondria. Furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (GRNs) for IRG1 in mouse and human macrophages. We further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of IRG1. Among the computationally identified regulators, siRNA-mediated gene silencing of interferon regulatory factor 1 (IRF1) in macrophages significantly decreased the expression of IRG1/CAD at the gene and protein level, which correlated with a reduced production of itaconic acid. Using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of IRG1 expression and could pave the way to therapeutic approaches targeting itaconic acid levels.

Inflammation Promotes a Conversion of Astrocytes into Neural Progenitor Cells via NF-κB Activation

Brain inflammation, a common feature in neurodegenerative diseases, is a complex series of events, which can be detrimental and even lead to neuronal death. Nonetheless, several studies suggest that inflammatory signals are also positively influencing neural cell proliferation, survival, migration, and differentiation. Recently, correlative studies suggested that astrocytes are able to dedifferentiate upon injury and may thereby re-acquire neural stem cell (NSC) potential. However, the mechanism underlying this dedifferentiation process upon injury remains unclear. Here, we report that during the early response of reactive gliosis, inflammation induces a conversion of mature astrocytes into neural progenitors. A TNF treatment induces the decrease of specific astrocyte markers, such as glial fibrillary acidic protein (GFAP) or genes related to glycogen metabolism, while a subset of these cells re-expresses immaturity markers, such as CD44, Musashi-1, and Oct4. Thus, TNF treatment results in the appearance of cells that exhibit a neural progenitor phenotype and are able to proliferate and differentiate into neurons and/or astrocytes. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we highlight a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-κB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-κB pathway.