Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech.

Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1+ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA-SET2 acting as an alarmin-like molecule whose role is to locally transmit a "danger" signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response.

Effects of Carbon Nanotube Environmental Dispersion on an Aquatic Invertebrate, Hirudo medicinalis.

The recent widespread applications of nanomaterials, because of their properties, opens new scenarios that affect their dispersal in the environment. In particular multiwall carbon nanotubes (MWCNTs), despite their qualities, seem to be harmful for animals and humans. To evaluate possible toxic effects caused by carbon nanotube environmental dispersion, with regard to aquatic compartment, we proposed as experimental model a freshwater invertebrate: Hirudo medicinalis. In the present study we analyse acute and chronic immune responses over a short (1, 3, 6 and 12 hours) and long time (from 1 to 5 weeks) exposure to MWCNTs by optical, electron and immunohistochemical approaches. In the exposed leeches angiogenesis and fibroplasia accompanied by massive cellular migration occur. Immunocytochemical characterization using specific markers shows that in these inflammatory processes the monocyte-macrophages (CD45+, CD68+) are the most involved cells. These immunocompetent cells are characterized by sequence of events starting from the expression of pro-inflammatory cytokines (in particular IL-18), and amyloidogenensis. Our combined experimental approaches, basing on high sensitive inflammatory response can highlight adverse effects of nanomaterials on aquatic organisms and could be useful to assess the MWCNTs impact on aquatic, terrestrial animal and human health.

Rational design of stable and functional hirudin III mutants with lower antigenicity.

Hirudin is an inhibitor of thrombin and used as an effective anticoagulant, but has a potential to develop unacceptable immune responses. In this study, two computational tools were used to predict T-cell epitopes within Hirudin variant III (HVIII) sequence, and design mutations that would lessen its antigenicity. Homology models of native and mutant HVIII proteins (T4K, S9G, V21G, and V21K) were generated, and further used to assess their interactions with thrombin. The docking experiment showed that all mutants had a suitable pattern of interactions, with similar or lower interaction energies compared with the native protein. These complexes were subsequently subjected to molecular dynamics simulation. All mutants complexes had overall stable structures over simulation time, with RMSD, gyration radius, hydrogen bonds numbers, and accessible surface areas patterns that were comparable with the native HVIII over time. Interestingly, in all mutants, a shorter length was observed for the two salt bridges Arg73-Asp55 and Arg77-Glu57, which are suggested to be important in Hirudin-thrombin complex formation. Best selected mutants expressed in Escherichia coli BL21(DE3), subsequently SDS-PAGE and Western blot analysis confirmed the successful same expression of Hirudin and mutants. In conclusion, we believe that this computational approach could identify potentially safer proteins with preserved or even improved functionality.

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.

Construction of a medicinal leech transcriptome database and its application to the identification of leech homologs of neural and innate immune genes.

BACKGROUND: The medicinal leech, Hirudo medicinalis, is an important model system for the study of nervous system structure, function, development, regeneration and repair. It is also a unique species in being presently approved for use in medical procedures, such as clearing of pooled blood following certain surgical procedures. It is a current, and potentially also future, source of medically useful molecular factors, such as anticoagulants and antibacterial peptides, which may have evolved as a result of its parasitizing large mammals, including humans. Despite the broad focus of research on this system, little has been done at the genomic or transcriptomic levels and there is a paucity of openly available sequence data. To begin to address this problem, we constructed whole embryo and adult central nervous system (CNS) EST libraries and created a clustered sequence database of the Hirudo transcriptome that is available to the scientific community. RESULTS: A total of approximately 133,000 EST clones from two directionally-cloned cDNA libraries, one constructed from mRNA derived from whole embryos at several developmental stages and the other from adult CNS cords, were sequenced in one or both directions by three different groups: Genoscope (French National Sequencing Center), the University of Iowa Sequencing Facility and the DOE Joint Genome Institute. These were assembled using the phrap software package into 31,232 unique contigs and singletons, with an average length of 827 nt. The assembled transcripts were then translated in all six frames and compared to proteins in NCBI's non-redundant (NR) and to the Gene Ontology (GO) protein sequence databases, resulting in 15,565 matches to 11,236 proteins in NR and 13,935 matches to 8,073 proteins in GO. Searching the database for transcripts of genes homologous to those thought to be involved in the innate immune responses of vertebrates and other invertebrates yielded a set of nearly one hundred evolutionarily conserved sequences, representing all known pathways involved in these important functions. CONCLUSIONS: The sequences obtained for Hirudo transcripts represent the first major database of genes expressed in this important model system. Comparison of translated open reading frames (ORFs) with the other openly available leech datasets, the genome and transcriptome of Helobdella robusta, shows an average identity at the amino acid level of 58% in matched sequences. Interestingly, comparison with other available Lophotrochozoans shows similar high levels of amino acid identity, where sequences match, for example, 64% with Capitella capitata (a polychaete) and 56% with Aplysia californica (a mollusk), as well as 58% with Schistosoma mansoni (a platyhelminth). Phylogenetic comparisons of putative Hirudo innate immune response genes present within the Hirudo transcriptome database herein described show a strong resemblance to the corresponding mammalian genes, indicating that this important physiological response may have older origins than what has been previously proposed.

Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in the lesioned central nervous system using a leech model.

A highly conserved ortholog of the human complex p43/endothelial monocyte-activating polypeptide II (EMAPII) was characterized in the CNS of the leech Hirudo medicinalis. As observed in mammals, the leech complex is processed to release the cytokine HmEMAPII. Taking advantages of these similarities, we have attempted to elucidate the role of EMAPII in the CNS using the leech model. Although EMAPII is considered a modulator of inflammatory reactions within the peripheral innate immune response in humans, its function in CNS immunity has yet to be described. Chemotaxis assays were conducted, revealing the ability of EMAPII to exert a chemoattractant effect on both leech and human microglial cells, indicating a novel function of this cytokine in the human brain. Quantitative RT-PCR analysis together with in situ hybridization and immunohistochemistry approaches showed that bacterial challenge induced the expression of HmEMAPII at the lesion site where microglial cells accumulated. Moreover, gene silencing experiments have demonstrated that the gene expression of HmEMAPII is under the control of a signaling pathway associated with the TLR HmTLR1, newly characterized in the CNS of our model. To the best of our knowledge, this is the first report showing evidence for (1) the chemoattractant properties of EMAPII on leech and human microglial cells, (2) the regulation by a TLR of the expression of a gene encoding a cytokine in the CNS of an invertebrate, and (3) an immune function of a TLR in a lophotrochozoan model.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia.

Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair.