Role of RNASET2 in innate immune response regulation

Numerous studies have highlighted the existence of enzymes that, besides presenting a conserved role, take part in different biological processes acquiring new functions. In particular, the T2 RNase family of enzymes is one of the most investigated group, which is involved not only in RNA regulation, but also performs additional functions, regardless of the catalytic activity. It has been recently observed that, through the continuous cross-talk between the cancer cells and the adjacent microenvironment and the activation of inflammatory response by inducing macrophages activation, the human protein RNASET2 is able to control the ovarian cancer malignancy. However, the details about the mechanisms by which T2 enzymes interact with immune system remain poorly defined. The medicinal leech Hirudo verbana is here proposed as a consolidated invertebrate model to shed light on these aspects. The results presented in this project reveal that both human and leech T2 RNases cause fibroplasia, connective tissue remodeling and macrophage recruitment in leeches. Moreover, in vivo and in vitro experiments proved that HvRNASET2, released by granulocytes after bacterial infection, is a candidate molecule for counteracting pathogens. In particular, it plays a dual role, inducing bacterial clustering to facilitate macrophage phagocytosis and promoting immunocompetent cells recognition. In conclusion, HvRNASET2 acts as an effector of innate immune response, to favore an efficient microbial pathogen elimination and an effective oncosuppressive activity. These studies, related to roles of T2 RNases in tissue remodeling, immune response modulation and antimicrobial activity, could be essential for developing new therapeutic application

Invertebrate Models in Innate Immunity and Tissue Remodeling Research

Abstract is not provided for the editoria

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics

Introduction:In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods:The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion:The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture

Role of RNASET2 in innate immune response regulation

Numerous studies have highlighted the existence of enzymes that, besides presenting a conserved role, take part in different biological processes acquiring new functions. In particular, the T2 RNase family of enzymes is one of the most investigated group, which is involved not only in RNA regulation, but also performs additional functions, regardless of the catalytic activity. It has been recently observed that, through the continuous cross-talk between the cancer cells and the adjacent microenvironment and the activation of inflammatory response by inducing macrophages activation, the human protein RNASET2 is able to control the ovarian cancer malignancy. However, the details about the mechanisms by which T2 enzymes interact with immune system remain poorly defined. The medicinal leech Hirudo verbana is here proposed as a consolidated invertebrate model to shed light on these aspects. The results presented in this project reveal that both human and leech T2 RNases cause fibroplasia, connective tissue remodeling and macrophage recruitment in leeches. Moreover, in vivo and in vitro experiments proved that HvRNASET2, released by granulocytes after bacterial infection, is a candidate molecule for counteracting pathogens. In particular, it plays a dual role, inducing bacterial clustering to facilitate macrophage phagocytosis and promoting immunocompetent cells recognition. In conclusion, HvRNASET2 acts as an effector of innate immune response, to favore an efficient microbial pathogen elimination and an effective oncosuppressive activity. These studies, related to roles of T2 RNases in tissue remodeling, immune response modulation and antimicrobial activity, could be essential for developing new therapeutic application

Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech <i>Hirudo verbana</i>

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation

Inflammation events occurring upon bacterial infection in Mytilus galloprovincialis

Bivalves, and in particular the Mediterranean Mytilus galloprovincialis are important sources of food in several countries in the world. Because of that, mussels farming has a strong economic impact. Due to their status as sessile and filter-feeding animals, bivalves accumulate in their tissues environmental pollutants and a larger amount of microorganisms and between these, a multitude of infective bacteria for higher vertebrates and humans, such as Vibrio species. Several immunological responses of M. galloprovincialis were investigated and described after Vibrio infection both, in vitro and in vivo conditions, such as hemocytes count and different cellular subpopulations. Particularly, intracellular signaling pathways are activated to trigger the synthesis of antimicrobial effectors Here, were investigated the modulation of immunological cellular markers of the Mediterranean bivalve M. galloprovincialis in response to in vivo exposure with Vibrio splendidus. The activation of inflammatory cascade was examined through immunolabeling with antibodies involved in the pathway: Toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MYD88), Allograft inflammatory factor-1 (AIF1) and ribonucleases RNASET2 (T2 family), that trigger the recruitment and activation of macrophages in vertebrates. Results confirmed the activation of TRL4 during bacterial infection and MYD88 adapter suggesting a role in recognition and intracellular signaling. Moreover, Gram-negative bacteria determine the recruitment by the ribonuclease RNASET2 of haemocytes and a huge migration of AIF-1+ cells. This approach is suitable to understand the molecular defense mechanisms in invertebrates during the exposure to possible pathogens, also in order to develop new technics and tools to evaluate mussel immunity response used in aquaculture to prevent mass mortality of these mollusks, economic loss and potential risks for consumers of seafood

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech&rsquo;s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health

A new cellular type in invertebrates: first evidence of telocytes in leech Hirudo medicinalis

Abstract Telocytes, a peculiar cell type, were recently found in vertebrates. Hence this cell system has been reported as ubiquitous in the bodies of mammals and interpreted as an important player in innate immunity and tissue regeneration, it is reasonable to look for it also in invertebrates, that rely their integrity solely by innate immunity. Here we describe, at morphological and functional level, invertebrate telocytes from the body of leech Hirudo medicinalis (Annelida), suggesting how these cells, forming a resident stromal 3D network, can influence or participate in different events. These findings support the concepts that leech telocytes: i) are organized in a cellular dynamic and versatile 3D network likewise the vertebrate counterpart; ii) are an evolutionarily conserved immune-neuroendocrine system; iii) form an immuno-surveillance system of resident cells responding faster than migrating immunocytes recruited in stimulated area; iv) communicate with neighbouring cells directly and indirectly, via cell-cell contacts and soluble molecules secreted by multivesicular bodies; v) present within neo-vessels, share with immunocytes the mesodermal lineage; vi) are involved in regenerative processes. In conclusion, we propose that HmTCs, integrating so different functions, might explain the innate immune memory and can be associated with several aged related diseases

Step-by-Step Regeneration of Tentacles after Injury in Anemonia viridis—Morphological and Structural Cell Analyses

Benthic marine invertebrates, such as corals, are often subjected to injury caused by several sources. Here, the differences and characteristics in injured and health tissues in terms of cellular components are shown through a histological investigation of the soft coral Anemonia viridis at 0 h, 6 h, 24 h, and 7 days after injury caused by tentacle amputation. In addition, a new tool was used for the first time in invertebrates, positron emission tomography, in order to investigate the events that occur during regeneration within a longer time period (0 h, 24 h, and 14 days after the tentacles were cut). Higher integrated density values were measured through a densitometric analysis in sections stained with Fontana–Masson at 24 h after the tentacles were cut. This suggests an increase in melanin-like containing cells and a subsequent increase in fibroblast-like cells differentiated by amoebocytes that converge to the lesion site in the early stages of inflammation and regeneration. This work provides, for the first time, an elucidation of the events that occur during wound-healing and regeneration in basal metazoan, focusing on the characterisation of immune cells and their role. Our results indicate that Mediterranean anthozoan proves to be a valuable model for studying regeneration. Many events highlighted in this research occur in different phyla, suggesting that they are highly conserved

Acid phosphatase reaction (A-F) and anti CD68 immunofluorescence staining (G-L).

<p>Resident in untreated (A, G) and migrating macrophages-like cells in treated leeches (B-E) and (H-L), located under the epithelium (e) and among the muscle fibers (m), are positive for acid phosphatase reaction (arrowheads in A-F) and for anti-CD68 (G-L). (F) Quantitative evaluation of cell numbers. Column 1: number of cells in untreated sample, columns 2–10: number of cells in MWCNTs treated sample from 1h up to 5 weeks. *p<0.01. (J) Combined transmission and fluorescence images showing CD68⁺ cells (in red) in spatial association with MWCNTs aggregates (circled). Bars in A-E, G-I, K-L: 100μm; Bar in F, J: 10μm.</p