IFI16 phase separation via multi-phosphorylation drives innate immune signaling.

The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3ß, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.

Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes.

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy-healthy, healthy-diseased, and diseased-diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Toll-like receptor 2 and α-Smooth Muscle Actin expressed in the tunica of a urochordate, Styela plicata.

The tunicate, Styela plicata (Lesueur, 1823) present an open circulator system with a tubular heart and blood flowing in lacunae among organs, bathing the tissues directly. Blood vascular lacunae are present in the tunica that is situated outside the epidermis and present a fibrous structure. The cells of the tunic are in straight contact with the blood vessels or are highly mobile. Ascidians are considered model organisms in comparative immunology of the chordate, and hold an important phylogenetic position as sister group of vertebrates. In recent years, numerous studies have reported the presence of Toll-like receptors (TLRs) in the genome of non-mammalian organisms including invertebrates. Two TLRs, designated Ci-TLR1 and Ci-TLR2 were expressed in the stomach, intestine and in numerous hemocytes of Ciona intestinalis, demonstrating that these key transmembrane proteins are evolutionarily conserved in ascidians. In this study for the first time, hemocytes aggregates were identified by confocal immunofluorescence techniques, using TLR2 antibody in the tunica of Styela plicata; furthermore, α-Smooth Muscle Actin (α-SMA) expression has been shown in the cells lining the vessels of the tunic. Our results support the view that the TLR-mediated innate immune functions are conserved in ascidian tissues.

Characterisation and functional role of a novel C1qDC protein from a colonial ascidian.

As an invertebrate, the compound ascidian Botryllus schlosseri faces nonself only with innate immunity. In this species, we already identified the key components of the lectin and alternative complement activation pathways. In the present work, by mining the transcriptome, we identified a single transcript codifying for a protein, member of the C1q-domain-containing protein family, with a signal peptide followed by two globular C1q (gC1q) domains. It shares a similar domain organisation with C1q/TNF-related proteins 4, the only vertebrate protein family with two gC1q domains. Our gC1q domain-containing protein, called BsC1qDC, is actively transcribed by immunocytes. The transcription is modulated during the Botryllus blastogenetic cycle and is upregulated following the injection of Bacillus clausii cells in the circulation. Furthermore, the injection of bsc1qdc iRNA in the vasculature results in decreased transcription of the gene and a significant impairment of phagocytosis and degranulation, suggesting the involvement of this molecule in immune responses.

Novel insights about the ascidian dopamine system: Pharmacology and phylogenetics of catecholaminergic receptors on the Phallusia nigra immune cells.

Dopamine (DA) is an important molecule that plays a role in the nervous and immune systems. DA is produced by a wide variety of animals and it is considered one of the oldest neurotransmitters. However, its specific function in immune cells has not been completely revealed. In a group of chordate animals, the ascidians, DA is reported to be produced by cells in the central nervous system (CNS); however, no dopaminergic receptor in their genomes has been described until now. Because this is an integrating characteristic of the ascidian dopamine system, here it was investigated the pharmacology, function, and phylogeny of DA and dopaminergic receptors (DRs) in the modulation of nitric oxide (NO) in the Phallusia nigra immune cells. The data disclosed, for the first time, that DA modulates NO production by immune cells. Its modulation probably occurs though adrenergic receptors, which display a special characteristic, in that they are capable of binding to noradrenaline (NA) and DA. A pharmacological analysis revealed that receptors present on the ascidian immune cells showed a high affinity to butaclamol, a non-selective D2-class receptor, increasing NO production. In addition, calcium intracellular mobilization was observed when DA was added to immune cells. In conclusion, the data revealed novel insights about the presence of catecholaminergic receptors (CRs) on the P. nigra immune cells, indicating that ascidian CRs have special pharmacological characteristics that are worth highlighting from an evolutionary point of view.

BsTLR1: A new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.

Toll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defence. As regards TLRs, two transcripts have been described and characterised in the solitary species Ciona intestinalis, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we mined our available transcriptome of the colonial ascidian Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR1, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. It is actively transcribed by both phagocytes and morula cells, the two circulating immunocyte types. In addition, we analysed bstlr1 transcription in vivo and in vitro, in different phases of the Botryllus blastogenetic cycle and under various experimental conditions. Our data show that there is a change in gene expression and mRNA location, according to the blastogenetic phase. Furthermore, we used a commercial antibody raised against the ectodomain of hTLR5 to study the possible functional role of Botryllus TLR(s). We observed that anti-hTLR5 significantly decreased in vitro phagocytosis and morula cell degranulation, two typical responses to the recognition of nonself. Collectively, our data add new information on the mechanisms of nonself recognition in a colonial ascidian.

Antitumoral compounds from vertebrate sister group: A review of Mediterranean ascidians.

Among the diseases that afflict the human population, cancer is one for which many drug treatments are not yet known or effective. Moreover, the pharmacological treatments used often create serious side effects in sick patients and for this reason, it is essential to find effective and less harmful treatments. To date, marine biodiversity is a real source of metabolites with antitumoral activity and among invertebrates' ascidians have been the main source to obtain them. Mediterranean area is the richest in biodiversity and contains several ascidian species used in drugs development during the years. However, many more Mediterranean ascidian species have not been studied and could be a source of useful bioactive compounds. This review aims to summarize the scientific studies that analyzed the antitumor compounds obtained from different Mediterranean ascidians species, encouraging them to search further compounds in other new species to improve pharmacological treatments and human population life.

Oxidative stress: Noradrenaline as an integrator of responses in the neuroendocrine and immune systems of the ascidian Phallusia nigra.

Neurotransmitters play key roles in regulating the homeostasis of organisms in stressful environments. Noradrenaline (NA) is the main neurotransmitter known to modulate immunological parameters, and is important in the crosstalk between the neuroendocrine and immune systems. In this study, using the ascidian Phallusia nigra, we analyzed the level of catecholamines (CA) in the plasma after mechanical stress, and the effect of NA on the oxidative stress (OS) displayed by immune cells. We measured the concentration of reactive oxygen species (ROS), and analyzed whether α- and/or ß-adrenoreceptors (ARs) are involved in ROS modulation, lipid peroxidation (LPO), antioxidant capacity against peroxyl radicals (ACAP), and activity of the enzymes catalase (CAT) and glutathione S transferase (GST) in immune cells after incubation with different concentrations of NA, with or without zymosan (ZnA) challenge. The results showed that NA reduced ROS production, even in immune cells challenged with ZnA, and that this modulation occurred through α1-and ß1-ARs. ACAP levels showed different responses, depending on whether immune cells were challenged or not with ZnA, and also depending on the NA concentration: 1.0 µM NA increased ACAP levels, but 10.0 µM reduced ACAP levels. NA enhanced the activity of CAT and GST in ZnA-challenged and non-challenged immune cells, while 1.0 and 10.0 µM NA effectively reduced LPO. Taken together, these results show that NA can protect cells from ROS damage, decreasing ROS production and LPO, and enhancing ACAP as well as the activity of CAT and GST. The approach used here with this model contributes to understanding the relationship between the neuroendocrine and immune systems, revealing new effects of NA on OS regulation in ascidians.

Evolutionary perspective on the hematopoietic system through a colonial chordate: allogeneic immunity and hematopoiesis.

Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition.

Complement system and phagocytosis in a colonial protochordate.

In the present work, we investigated, in the colonial ascidian Botryllus schlosseri, the role of complement C3 (BsC3) in phagocytosis. We studied the modulation of BsC3 transcription in the course of the colonial blastogenetic cycle, with particular reference to the takeover, when apoptotic cells in the tissues of old zooids are cleared by circulating phagocytes. In situ hybridisation with BsC3 riboprobes labelled only morula cells, the most abundant haemocytes. Anti-hC3 antibody recognised morula cells and also phagocytes when haemocytes were previously incubated with zymosan. The inhibition of C3 activation prevented the labelling of phagocytes. In phagocytosis assays with haemocytes from colonies injected with anti-hC3 antibody or bsc3 iRNA, the capability to ingest target cells was significantly (p < 0.001) reduced. Therefore, our results strongly support a key role of BsC3 in phagocytosis and open to new investigations on the nature of the receptors of the products of BsC3 activation.

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.

Complex mammalian-like haematopoietic system found in a colonial chordate.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Immunological response to bacterial infection in a pelagic tunicate: Inflammation in the salp Thalia democratica.

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells). We aimed to provide the first investigation of defense reactions upon exposure to a large amount of bacteria (Bacillus clausii). We detected (i) bacterial phagocytosis by hyaline amebocytes, (ii) degradation of phagocytizing hyaline amebocytes in the tunic after transcellular diapedesis from the hemocoel, and (iii) release of heparin, histamine, and TNF-α by granular cells. Cell degranulation and phagocytosis occurred in epidermal cells lining the hemocoel, and an excess of mucus was observed in the post-branchial gut, causing a functional inhibition of cilia and microvilli. These findings indicate multi-step events comparable to an inflammation involving responses at both tissue and organismal levels.

The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses.

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised.

Exploration of the Innate Immune System of Styela clava: Zn2+ Binding Enhances the Antimicrobial Activity of the Tunicate Peptide Clavanin A.

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, ß-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.

Balancing selection on allorecognition genes in the colonial ascidian Botryllus schlosseri.

Allorecognition is the capability of an organism to recognize its own or related tissues. The colonial ascidian Botryllus schlosseri, which comprises five genetically distinct and divergent species (Clades A-E), contains two adjacent genes that control allorecognition: fuhcsec and fuhctm. These genes have been characterized extensively in Clade A and are highly polymorphic. Using alleles from 10 populations across the range of Clade A, we investigated the type and strength of selection maintaining this variation. Both fuhc genes exhibit higher within-population variation and lower population differentiation measures (FST) than neutral loci. The fuhc genes contain a substantial number of codons with >95% posterior probability of dN/dS > 1. fuhcsec and fuhctm also have polymorphisms shared between Clade A and Clade E that were present prior to speciation (trans-species polymorphisms). These results provide robust evidence that the fuhc genes are evolving under balancing selection.

Developmental cell death programs license cytotoxic cells to eliminate histocompatible partners.

In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 × 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor.

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Botryllus schlosseri allorecognition: tackling the enigma.

Allorecognition has been well-studied in the context of vertebrate adaptive immunity and recognition of the Major Histocompatibility Complex (MHC), which is the central event of vertebrate immune responses. Although allorecognition systems have been identified throughout the metazoa, recent results have shown that there is no apparent conservation or orthologous relationship between the mechanisms underlying this phenomenon in different organisms. Thus the origin of the vertebrate adaptive immune system as well as these other complex recognition systems is a complete mystery. This review will focus on allorecognition in Botryllus schlosseri, a basal chordate which undergoes a natural transplantation reaction following contact between two individuals, and, analogous to vertebrates, is controlled by a single locus. We will summarize each of the known candidate genes within this locus and their potential roles in allorecognition, and speculate on how these findings may in fact be revealing potential functional relationships between disparate allorecognition systems.

Histamine regulates the inflammatory response of the tunicate Styela plicata.

Histamine is stored inside hemocytes of the tunicate Styela plicata (Chordata, Tunicata, Ascidiacea), but no evidence on its role in the regulation of the immune response of this species has been reported. We examined whether histamine participated in the regulation of inflammation and host defense in S. plicata. The presence of histamine inside S. plicata hemocytes was confirmed by flow cytometry, and histamine release was detected by ELISA, after in vitro hemocyte stimulation with different PAMPs. In vitro hemocyte treatment with histamine, or specific histamine-receptor agonists, reduced their phagocytic ability. Injection of histamine into the tunic recruited hemocytes to the site of injection. Systemic injection of histamine, or the histamine-releasing agent compound 48/80, decreased the phagocytic ability of hemocytes. Histamine promoted the constriction of tunic hemolymph vessels in vivo, having a direct effect on vasoconstriction in tunic explants. These results provide for the first time clear evidence for the involvement of histamine in the regulation of inflammation and host defense in tunicates.