Pan-claudin family interactome analysis reveals shared and specific interactions.

Claudins are a family of transmembrane proteins expressed in epithelial tissues and are the major components of tight junctions (TJs), which define barrier properties in epithelia and maintain cell polarity. How claudins regulate the formation of TJs and which functions they exert outside of them is not entirely understood. Although the long and unstructured C-terminal tail is essential for regulation, it is unclear how it is involved in these functions beyond interacting with TJ-associated proteins such as TJ protein ZO-1 (TJP1). Here, we present an interactome study of the pan-claudin family in Madin-Darby canine kidney (MDCK)-C7 cells by combining two complementary mass spectrometry-based pull-down techniques creating an interaction landscape of the entire claudin family. The interaction partners of the claudins' C termini reveal their possible implications in localized biological processes in epithelial cells and their regulation by post-translational modifications (PTMs).

New insights into the organization and regulation of the apical polarity network in mammalian epithelial cells.

Cell polarity is a fundamental property of most animal cells and is critical during development and for most cell and tissue functions. Epithelial cells are organized into apical and basolateral compartments, and this intrinsic cellular asymmetry is essential for all functions that are carried out by epithelial tissue. The establishment of a polarized epithelial phenotype is orchestrated by major rearrangements of the cell cytoskeleton, polarized membrane trafficking, the formation and maturation of epithelial cell junctions, cell signaling pathways, and the generation of cortical phospholipid asymmetry. These processes need to be coordinated precisely in time and space and integrated with physical and chemical signals from the environment, failure of which leads to severe developmental disorders and various human diseases. At the heart of this regulatory network are the evolutionarily conserved polarity modules Par, Crumbs, and Scribble, whose components engage in complex cooperative and antagonistic interactions to compartmentalize and functionalize the epithelial cell cortex and to control the spatiotemporal activity of downstream polarity effectors. In this review, we will discuss recent insights into the organization and regulation of the mammalian Par and Crumbs modules and outline a hypothetical framework of how these proteins orchestrate epithelial polarity development, HIPPO signaling, and actomyosin activity at the apical-lateral border.

Nanomaterials and Annelid Immunity: A Comparative Survey to Reveal the Common Stress and Defense Responses of Two Sentinel Species to Nanomaterials in the Environment.

Earthworms and leeches are sentinel animals that represent the annelid phylum within terrestrial and freshwater ecosystems, respectively. One early stress signal in these organisms is related to innate immunity, but how nanomaterials affect it is poorly characterized. In this survey, we compare the latest literature on earthworm and leeches with examples of their molecular/cellular responses to inorganic (silver nanoparticles) and organic (carbon nanotubes) nanomaterials. A special focus is placed on the role of annelid immunocytes in the evolutionarily conserved antioxidant and immune mechanisms and protein corona formation and probable endocytosis pathways involved in nanomaterial uptake. Our summary helps to realize why these environmental sentinels are beneficial to study the potential detrimental effects of nanomaterials.

Aphidius ervi Teratocytes Release Enolase and Fatty Acid Binding Protein Through Exosomal Vesicles.

The molecular bases of the host-parasitoid interactions in the biological system Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) and Aphidius ervi (Haliday) (Hymenoptera, Braconidae) have been elucidated allowing the identification of a gamma-glutamyl transpeptidase, the active component of maternal venom secretion, and teratocytes, the embryonic parasitic factors responsible for host physiology regulation after parasitization. Teratocytes, cells deriving from the dissociation of the serosa, the parasitoid embryonic membrane, are responsible for extra-oral digestion of host tissues in order to provide a suitable nutritional environment for the development of parasitoid larvae. Teratocytes rapidly grow in size without undergoing any cell division, synthesize, and release in the host hemolymph two proteins: a fatty acid binding protein (Ae-FABP) and an enolase (Ae-ENO). Ae-FABP is involved in transport of fatty acids deriving from host tissues to the parasitoid larva. Ae-ENO is an extracellular glycolytic enzyme that functions as a plasminogen like receptor inducing its activation to plasmin. Both Ae-FABP and Ae-ENO lack their signal peptides, and they are released in the extracellular environment through an unknown secretion pathway. Here, we investigated the unconventional mechanism by which teratocytes release Ae-FABP and Ae-ENO in the extracellular space. Our results, obtained using immunogold staining coupled with TEM and western blot analyses, show that these two proteins are localized in vesicles released by teratocytes. The specific dimension of these vesicles and the immunodetection of ALIX and HSP70, two exosome markers, strongly support the hypothesis that these vesicles are exosomes.

The medicinal leech as a valuable model for better understanding the role of a TLR4-like receptor in the inflammatory process.

Despite extensive investigation focused on both the molecular characteristics and the expression level of Toll-like receptors (TLRs) during the inflammatory response in vertebrates, few data are available in the literature on the role of these proteins in invertebrate's immune response. Here, we propose the medicinal leech as a valuable model to better elucidate the role of TLR4 and its related products, such as tumor necrosis factor (TNF-α), after activation of the leech peripheral immune system with the endogenous medicinal leech recombinant allograft inflammatory factor-1 (rHmAIF-1) or with an exogenous stimulus, such as lipopolysaccharide (LPS). Our results indicate that activated macrophages (HmAIF-1+) and granulocytes (CD11b+) express both TLR4 and its coreceptor CD14. Moreover, functional studies performed by injecting a cyanobacterium selective TLR4 antagonist CyP demonstrated that only the TLR4 pathway was blocked, while the immune response caused by lipoteichoic acid (LTA) treatment is not affected. These results are consistent with literature on vertebrates, indicating that TLR4 functions as a LPS receptor while the recognition of LTA may involve other pathways.

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.

Functional amyloidogenesis in immunocytes from the colonial ascidian Botryllus schlosseri: Evolutionary perspective.

Cytotoxic morula cells (MCs) and phagocytes are the circulating immunocytes of the colonial ascidian Botryllus schlosseri: Both these cells can synthesise amyloid fibrils, supporting the idea that physiological amyloidogenesis is involved in inflammation and modulation of immune responses. Intriguingly, amyloid of B. schlosseri immunocytes is made of two different proteins. MCs, the first cells to sense non-self and involved in the allorejection reaction between contacting genetically incompatible colonies, use melanin encapsulation as the principal method to fight non-self. They release amyloid fibrils formed by p102 protein that allow the packaging and deposit of melanin and other toxic molecules nearby the invader or in the contact region of incompatible colonies. Phagocytes release amyloid-based extracellular traps when challenged with microbes: their amyloid fibrils harbour BsAPP, an orthologue of the vertebrate amyloidogeneic protein APP. This strategy of immune response, present also in human neutrophils, allows phagocytes to block and engulf bacteria and fungi.

Rapamycin and fasting sustain autophagy response activated by ischemia/reperfusion injury and promote retinal ganglion cell survival.

Autophagy, the cellular process responsible for degradation and recycling of cytoplasmic components through the autophagosomal-lysosomal pathway, is fundamental for neuronal homeostasis and its deregulation has been identified as a hallmark of neurodegeneration. Retinal hypoxic-ischemic events occur in several sight-treating disorders, such as central retinal artery occlusion, diabetic retinopathy, and glaucoma, leading to degeneration and loss of retinal ganglion cells. Here we analyzed the autophagic response in the retinas of mice subjected to ischemia induced by transient elevation of intraocular pressure, reporting a biphasic and reperfusion time-dependent modulation of the process. Ischemic insult triggered in the retina an acute induction of autophagy that lasted during the first hours of reperfusion. This early upregulation of the autophagic flux limited RGC death, as demonstrated by the increased neuronal loss observed in mice with genetic impairment of basal autophagy owing to heterozygous ablation of the autophagy-positive modulator Ambra1 (Ambra1+/gt). Upregulation of autophagy was exhausted 24 h after the ischemic event and reduced autophagosomal turnover was associated with build up of the autophagic substrate SQSTM-1/p62, decreased ATG12-ATG5 conjugate, ATG4 and BECN1/Beclin1 expression. Animal fasting or subchronic systemic treatment with rapamycin sustained and prolonged autophagy activation and improved RGC survival, providing proof of principle for autophagy induction as a potential therapeutic strategy in retinal neurodegenerative conditions associated with hypoxic/ischemic stresses.

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

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.

Cellular responses induced by multi-walled carbon nanotubes: in vivo and in vitro studies on the medicinal leech macrophages.

The core characteristics of multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology however, since their production and use is steadily increasing, their environmental dispersion could be potentially hazardous to animal and human health. For this reason, the identification of new methods and of reliable models to better understand MWCNT effects is essential. Here we propose the medicinal leech as an 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's macrophages. Here we will focus on the direct effects of MWCNTs on these cells by isolating and culturing leech's macrophages by means of the consolidated Matrigel technique, followed by MWCNT in vitro treatment. Our results indicate that MWCNT administration causes both the decrease of cell proliferation rate and the increase of the apoptotic rate. Furthermore, since oxidative stress is linked with inflammation, reactive oxygen species has been evaluated confirming that their production rate increases after MWCNT treatment. Our experimental approaches demonstrate the ability of MWCNTs inducing a powerful inflammatory response and confirm that the medicinal leech is a good alternative model to study the possible harmful effects of any nanomaterial.

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Systemic distribution of single-walled carbon nanotubes in a novel model: alteration of biochemical parameters, metabolic functions, liver accumulation, and inflammation in vivo.

The increasing use of carbon nanotubes (CNTs) in several industrial applications raises concerns on their potential toxicity due to factors such as tissue penetrance, small dimensions, and biopersistence. Using an in vivo model for CNT environmental exposure, mimicking CNT exposition at the workplace, we previously found that CNTs rapidly enter and disseminate in the organism, initially accumulating in the lungs and brain and later reaching the liver and kidneys via the bloodstream in CD1 mice. Here, we monitored and traced the accumulation of single-walled CNTs (SWCNTs), administered systemically in mice, in different organs and the subsequent biological responses. Using the novel in vivo model, MITO-Luc bioluminescence reporter mice, we found that SWCNTs induce systemic cell proliferation, indicating a dynamic response of cells of both bone marrow and the immune system. We then examined metabolic (water/food consumption and dejections), functional (serum enzymes), and morphological (organs and tissues) alterations in CD1 mice treated with SWCNTs, using metabolic cages, performing serum analyses, and applying histological, immunohistochemical, and ultrastructural (transmission electron microscopy) methods. We observed a transient accumulation of SWCNTs in the lungs, spleen, and kidneys of CD1 mice exposed to SWCNTs. A dose- and time-dependent accumulation was found in the liver, associated with increases in levels of aspartate aminotransferase, alanine aminotransferase and bilirubinemia, which are metabolic markers associated with liver damage. Our data suggest that hepatic accumulation of SWCNTs associated with liver damage results in an M1 macrophage-driven inflammation.

Amino acid transporter B(0)AT1 (slc6a19) and ancillary protein: impact on function.

Amino acids play an important role in the metabolism of all organisms. Their epithelial re-absorption is due to specific transport proteins, such as B(0)AT1, a Na(+)-coupled neutral amino acid symporter belonging to the solute carrier 6 family. Here, a recently cloned fish orthologue, from the intestine of Salmo salar, was electrophysiologically characterized with the two-electrode voltage clamp technique, in Xenopus laevis oocytes heterologously expressing the transporter. Substrate specificity, apparent affinities and the ionic dependence of the transport mechanism were determined in the presence of specific collectrin. Results demonstrated that like the human, but differently from sea bass (Dicentrarchus labrax) orthologue, salmon B(0)AT1 needs to be associated with partner proteins to be correctly expressed at the oocyte plasma membrane. Cloning of sea bass collectrin and comparison of membrane expression and functionality of the B(0)AT1 orthologue transporters allowed a deeper investigation on the role of their interactions. The parameters acquired by electrophysiological and immunolocalization experiments in the mammalian and fish transporters contributed to highlight the dynamic of relations and impacts on transport function of the ancillary proteins. The comparative characterization of the physiological parameters of amino acid transporters with auxiliary proteins can help the comprehension of the regulatory mechanism of essential nutrient absorption.

Extracellular matrix degradation via enolase/plasminogen interaction: Evidence for a mechanism conserved in Metazoa.

BACKGROUND INFORMATION: While enolase is a ubiquitous metalloenzyme involved in the glycolytic pathway, it is also known as a multifunctional protein, since enolases anchored on the outer surface of the plasma membrane are involved in tissue invasion. RESULTS: We have identified an extracellular enolase (Ae-ENO) produced by the teratocytes, embryonic cells of the insect parasitoid Aphidius ervi. We demonstrate that Ae-ENO, although lacking a signal peptide, accumulates in cytoplasmic vesicles oriented towards the cell membrane. Ae-ENO binds to and activates a plasminogen-like molecule inducing digestion of the host tissue and thereby ensuring successful parasitism. CONCLUSIONS: These results support the hypothesis that plasminogen-like proteins exist in invertebrates. Interestingly the activation of a plasminogen-like protein is mediated by a mechanisms involving the surface enolase/fibrinolytic system considered, until now, exclusive of vertebrates, and that instead is conserved across species. SIGNIFICANCE: To our knowledge, this is the first example of enolase mediated Plg-like binding and activation in insect cells, demonstrating the existence of an ECM degradation process via a Plg-like protein in invertebrates.

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.

The main actors involved in parasitization of Heliothis virescens larva.

At the moment of parasitization by another insect, the host Heliothis larva is able to defend itself by the activation of humoral and cellular defenses characterized by unusual reactions of hemocytes in response to external stimuli. Here, we have combined light and electron microscopy, staining reactions, and immunocytochemical characterization to analyze the activation and deactivation of one of the most important immune responses involved in invertebrates defense, i.e., melanin production and deposition. The insect host/parasitoid system is a good model to study these events. The activated granulocytes of the host insect are a major repository of amyloid fibrils forming a lattice in the cell. Subsequently, the exocytosed amyloid lattice constitutes the template for melanin deposition in the hemocel. Furthermore, cross-talk between immune and neuroendocrine systems mediated by hormones, cytokines, and neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropin hormone, alpha melanocyte-stimulating hormone, and neutral endopeptidase occurs. Thus, parasitization promotes massive morphological and physiological modifications in the host insect hemocytes and mimics general stress conditions in which phenomena such as amyloid fibril formation, melanin polymerization, pro-inflammatory cytokine production, and activation of the adrenocorticotropin hormone system occur. These events observed in invertebrates are also reported in the literature for vertebrates, suggesting that this network of mechanisms and responses is maintained throughout evolution.