Cellular responses induced by multi-walled carbon nanotubes: in vitro study on the medicinal leech macrophages

The intrinsic characteristics of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however their environmental dispersion could be potentially hazardous to animal and human health. Since the production and use of MWCNTs is steadily increasing, intentional or unintentional environmental discharges may occur. For this reason, the development of new methods and the identification of reliable models to completely understand MWCNTs effects are critical. Here we propose a freshwater invertebrate, the medicinal leech, as alternative model to assess the effects of MWCNTs on immune system. Our previous studies have already demonstrated that in vivo MWCNT treatment induces the activation of leech\u2019s macrophages. In this study, we focus on the direct effects of MWCNTs on these cells. We used the consolidated experimental approach, based on the injection in the leech body wall of the biomatrice Matrigel (MG) added with the cytokine Allograft inflammatory factor-1 (HmAIF-1), to specifically chemoattract macrophages. Cells extracted from MG were cultured and characterized with the specific markers CD45 and CD68, confirming their belonging to the monocyte-macrophage lineage. Primary macrophage cultures were then subjected to an in vitro treatment with MWCNTs at different concentrations (2.5, 5, 10, 25, 50 and 100 \u3bcg/ml). Our results indicate that leech macrophages, once in close contact with MWCNTs, actively produce amyloid material to encapsulate the foreign bodies. We also demonstrated that MWCNTs in vitro treatment cause the decrease of cell proliferation rate and the increase of the apoptotic rate. Furthermore, since oxidative stress is linked with inflammation and amyloid production, reactive oxygen species has been evaluated, confirming that their production rate increases after MWCNT treatment. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm the medicinal leech as a good alternative model that can be improved and successfully used to study the possible harmful effects of any nanomaterial. Moreover, since autophagic cell death pathway activation is emerging as a possible consequence of MWCNT treatment, in the future we will attempt to clarify this aspect in order to completely understand MWCNT-induces toxicity

TLRs EXPRESSION IN THE MEDICINAL LEECH

It is well known that Toll-like receptors (TLRs) play a central role for innate immunity in both in vertebrates and many invertebrates, by recognizing conserved pathogen-associated molecular patterns in order to trigger the innate immune response. Here, we evaluate the expression of TLR4, receptor of GRAM- bacterial lipopolysaccharides LPS, and of TLR2, receptor of GRAM+ lipoteichoic acid LTA after injection in the leech body wall of these two exogenous molecules known to induce a strong inflammatory response. Our results indicate that immune cells migrating toward the body wall of treated animals are macrophages (HmAIF-1+) and granulocytes (CD11b+) and express both TLR4 and TLR2. Moreover, the expression profile of TLR4 and TLR2 has been also evaluated by Western blot analysis highlighting the presence of immunoreactive products at about 130 and 110 kDa (for TLR4) and at about 100 kDa (for TLR2). We hypothesize that, according to literature, the presence of different molecular masses of TLR4 probably depends on its glycosylation state. Functional studies both in vivo and in vitro carried out by using CyP, a cyanobacterium selective TLR4 antagonist, were performed to inhibit the secretion of the pro-inflammatory cytokines, such as tumor necrosis factor alpha. Strikingly, stimulating with CyP and LPS or LTA, we found that only TLR4 pathway was blocked, while the immune response caused by LTA treatment is not affected. Our results, which are consistent with literature on vertebrates, indicate that TLR4 functions as LPS receptor while the recognition of gram-positive bacterial products involves TLR2. Further research will better clarify TLR4 and TLR2 expression and signalling in the medicinal leech

USE OF THE ANIMAL MODEL HIRUDO VERBANA (LEECH) TO EVALUATE THE POSSIBLE CORRELATION BETWEEN IMMUNE RESPONSE, AMYOLOIDOGENESIS AND CHRONIC INFLAMMATION

Toll-like receptors, essential pattern-recognition receptors (PRRs) of the innate immune system, recognize a range of conserved molecules of invading pathogens. Among them, TLR4 is expressed on the cell surface on both hematopoietic and non-hematopoietic cells, including cells of the central nervous system, playing a crucial role in both innate and neuroimmune responses. It is the receptor of LPS (Lipopolysaccharide) endotoxins, the major outer membrane components of Gram- bacteria and a potent activator of innate immunity and inflammatory response, inducing the expression of the proinflammatory molecules IL-18 and TNF-\u3b1. Recently, amyloidogenesis has been identified as new player in innate immune responses and has been proposed as a detoxifying event to fight ROS (reactive oxygen species) increase, given that an excessive oxidative stress becomes harmful to cells when their antioxidant capacities result insufficient to retain the appropriate redox state. Thus, an uncontrolled activation of the innate immune system can lead to amyloid fibrils accumulation and chronic inflammation. In addition, it has been shown that LPS and TLR4 are associated with Alzheimer disease (AD), characterized by the accumulation of amyloid fibrils and neuroinflammation. Within this context, microglia in the brain and monocytes/neutrophils in the periphery have a prominent role in initiating and regulating inflammation processes. Here we propose the use of the medicinal leech, Hirudo verbana, as a powerful model system to understand the role of TLR4 in innate immune response and neuroimmune activation. The advantages of using this model is found in its innate immune system that is very similar to Vertebrate\u2019s one, but lacking the complex cross-talk typical of adaptive immunity. Our in vivo and in vitro approaches, by means of histological, ultrastructural, immunohistochemical and Western Blot techniques aim to correlate amyloid fibrils and ROS production with LPS treatment, clarifying the relationship between peripheral and central nervous system immune responses. Furthermore, by blocking TLR4 intracellular cascade we demonstrate that both macrophages and microglia cells undergo to a rescue process that implicate amyloid fibrils degradation and restoration of physiological conditions. In conclusion, our study is promising to gain novel insight about the correlation between peripheral/neuro inflammation and amyloid accumulation

Allorecognition in the ascidian Botryllus schlosseri: the importance of amyloid

In the compound ascidian Botryllus schlosseri, allorecognition manifests primarily as colony specificity, controlled by a highly polymorphic Fu/HC locus, so that contacting colonies sharing at least one allele at the Fu/HC locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammation reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (MCs), inside the tips of the ampullae (the blind termini of the tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation with the consequent release of factors inducing autocrine and paracrine cell and the formation of necrotic, melanic spots (points of rejection; PORs) along the contact border. MCs are the first cells to sense nonself and are the source of cytokines that induce the recruitment of immunocytes at the inflammatory sites and the activation of phagocytes required for the clearance of foreign material. MCs store quinones, polyphenols and the enzyme phenoloxidase (PO) inside their granules, representing their cytotoxic potential. Although PO, quinones and polyphenols are soluble factors, it is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of the enzyme and the products of its activity is, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of degranulating MCs. With this idea in mind, we looked for factors released by MCs that could limit the spreading of cytotoxicity and melanisation. We found that MCs contain amyloid inside their granules and that amyloid fibrils are released in the surrounding medium upon MC degranulation forming a net-like scaffold entrapping PO and melanin, thus limiting their diffusion. In addition, the search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two Botryllus immunocyte types, i.e., phagocytes and MCs. This work represents the first demonstration of a physiological role of amyloid in protochordata immunity

The medicinal leech as a useful tool for studying haematopoiesis, angiogenesis and innate immune response

In the last decade, a large body of evidence has demonstrated that hematopoiesis, angiogenesis, immune response and cytokines regulating the occurrence of these processes have been highly conserved across Vertebrates and various invertebrate phyla. Here we provide a broad overview of medicinal leech hematopoiesis and we highlight the benefits of using the medicinal leech, having a simple anatomy and a repertoire of less varied cell types when compared to Vertebrates, as a powerful model for studying basic steps of hematopoiesis and immune responses. In particular, leeches\u2019 hematopoietic stem precursor cells (HSPCs), deriving from the activated botryoidal tissue, seem to share with their Vertebrates counterpart most morpho-functional and molecular mechanisms. HSPCs are massively recruited from the bloodstream towards sites of inflammation in response to injury, bacterial injection, or experimental administration of cytokines and contribute to immune response, neovascularization and muscle regeneration. Further, similar to Vertebrates these cells are driven toward a leukocyte, endothelial or myogenic, differentiation pathway, as a function of the time course of VEGF release to target cells. Since investigations on hematopoiesis can greatly help scientists and clinicians to better understand the pathological processes behind blood disorders, cancers and muscle regeneration, HSPCs of medicinal leech can be used as a powerful model system for understanding tissue stem cells and their role in angiogenesis, tissue regeneration and oncogenesis

The human recombinant RNASET2 activates the initial phase of the inflammatory response in the medicinal leech

Recent studies have demonstrated that RNASET2, the only member of the ribonuclease T2 family present in human genome, is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore its capacity to be a chemoattractant for numerous cells of monocytic-macrophage line and the possibility to act as an inducer of the innate immune response in Vertebrates have been established. In fact, in tumor tissues the detectable cross-talk between cancer cells and the surrounding tumor microenvironment is based on these aspects. Although several studies have been reported on the molecular features of RNASET2, the details on the pathways by which this evolutionarily conserved protein regulates the immune system are still poorly defined. In order to better elucidate these aspects, we report here the effect of the human recombinant RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. This animal has been chosen for its very simple anatomy and for its rapid inflammatory response. Indeed, after few hours from the injection, a large number of fibroblasts are visible in the connective tissue that appears completely remolded and infiltrated by numerous cells expressing the specifics macrophage markers CD68 and HmAIF1. In order to confirm the ability of this ribonuclease to attract macrophages, we used a consolidated experimental approach based on the injection in the leech body wall of the Matrigel biomatrice (MG), supplemented with the human recombinant RNASET2. After one week, the extracted MG sponges was infiltrated by numerous cells CD68+ and HmAIF1+. Moreover, in the leech body wall challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, endogenous RNASET2 is highly expressed by the numerous macrophages migrating to the site of inoculation. Taken together, these results clearly suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier