Francesco Drago

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium-binding adapter molecule 1 (Iba1/alias aif-1).

The Ionized calcium‐Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF‐1), is a 17 kDa cytokine‐inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF‐1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events.

Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68+ macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

The Leech Nervous System: A Valuable Model to Study the Microglia Involvement in Regenerative Processes

Microglia are intrinsic components of the central nervous system (CNS). During pathologies in mammals, inflammatory processes implicate the resident microglia and the infiltration of blood cells including macrophages. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects during neuropathological conditions in vivo and in vitro. The medicinal leech Hirudo medicinalis is a well-known model in neurobiology due to its ability to naturally repair its CNS following injury. Considering the low infiltration of blood cells in this process, the leech CNS is studied to specify the activation mechanisms of only resident microglial cells. The microglia recruitment is known to be essential for the usual sprouting of injured axons and does not require any other glial cells. The present review will describe the questions which are addressed to understand the nerve repair. They will discuss the implication of leech factors in the microglial accumulation, the identification of nerve cells producing these molecules, and the study of different microglial subsets. Those questions aim to better understand the mechanisms of microglial cell recruitment and their crosstalk with damaged neurons. The study of this dialog is necessary to elucidate the balance of the inflammation leading to the leech CNS repair.

ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs), which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs) propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012). However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

Calreticulin contributes to C1q-dependent recruitment of microglia in the leech Hirudo medicinalis following a CNS injury

Background The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech’s CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). Material/Methods Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. Results A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. Conclusions These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

Cytokine Impregnated Biomatrix: A New Tool to Study Multi-Wall Carbon Nanotubes Effects on Invertebrate Immune Cells

The novel features of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however they dissolved in water and accumulate in soils through the application of sewage sludge, accidental spills, and deposition from the air, agrochemicals or soil remediation. Given that several studies have revealed that chronic exposure to these nanomaterials products through the ingestion of drinking water, inhalation and dermal contact may harbour potential risks to human health, risk assessment of this nanomaterials in the aquatic environment are becoming essential. Here we propose a freshwater invertebrate, the leech Hirudo medicinalis, as a model to assess the effects MWCNTs on the immune system by means of in vivo and in vitro experiments. For this study, we used a consolidated experimental approach based on injection in the body wall of the leech of the biomatrice Matrigel (MG), added with a specific macrophage chemoattractant, the cytokine Allograft inflammatory factor-1 (AIF-1) and/or with MWCNTs. MG sponges analysis show the presence of a larger number of cells positive for both CD68 and HmAIF-1, specific monocyte-macrophage markers. Ultrastructural analysis suggests that MWCNTs may be internalized by phagocytosis but they seem also to be able to pierce cell membranes during cells migration. Cells extracted from MG were also used for in vitro treatment with MWCNTs at different concentration (2.5, 5, 10, 25, 50 and 100 μg/ml) for 24 h to study cell morphology changes and production of amyloid fibrils in order to encapsulate the foreign bodies. Our results, not only confirm the ability of MWCNTs in inducing a potent inflammatory response, but highlight rapid colorimetric assays that can be successfully used as sensitive tools for aquatic pollution biomonitoring.

Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus

Abstract Allograft inflammatory factor 1 (AIF‐1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF‐1 from the common sea urchin Paracentrotus lividus. In this species, AIF‐1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF‐1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF‐1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF‐1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF‐1 in the inflammatory response. Our analysis on P. lividus AIF‐1 contributes to elucidate AIF‐1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths. HighlightsA gene from Paracentrotus lividus encoding the Allograft inflammatory factor1 was identified.Expression of AIF‐1 was identified in coelomocytes and changed 24 h after immune challenge.The AIF‐1 protein showed different intracellular localization depending on the coelomocytes type.

Neuroprotection and Immunity in the Medicinal Leech Hirudo medicinalis : What About Microglia?

Abstract The central nervous system (CNS) of the medicinal leech ( Hirudo medicinalis ) is an immunocompetent organ, which surveys and responds to immune challenges through the production of antimicrobial molecules and phagocytic action. Similarly to vertebrates, leech microglia play a crucial role in CNS homeostasis under physiological and pathological conditions. The involvement of microglial cells in neuroprotection and inflammatory control upon bacterial challenges or CNS injury is currently poorly known. The alert mechanisms in the leech CNS activate microglia that modify their morphology and migrate to the challenged area. These activation events are linked to the production of chemotactic factors and the expression of inflammatory markers. This chapter gives an overview of immune response and nerve repair processes in the most studied invertebrate model for microglia.