Ascidians are tunicates, marine invertebrate chordates, considered the sister group of vertebrates ^[1][2][3][4][1,2,3,4]. They owe their name to the tunic, an epidermally secreted external layer that envelops the body. The tunic is composed of an ECM rich in collagen and tunicin (a form of cellulose) and also rich in immune cells ^{[5][6][7][8][9]}[5,6,7,8,9]. The tunic also contains proteins with 3,4-dihydroxyphenylalanine (DOPA), with a catechol moiety involved in the first line of immune reaction [10] and wound healing ^[11][12][11,12], and 3,4,5-trihydroxyphenylalanine (TOPA) with a pyrogallol moiety [13]. Ascidians are the source of many bioactive molecules belonging to a wide variety of chemical categories ^[14][15][14,15] and with potential health applications, including cytotoxic, antimitotic, antiviral and antimicrobial compounds ^{[16][17][18][19]}[16,17,18,19]. Most of the metabolites synthesized by ascidians contribute to creating the physico-chemical barrier preventing the entrance of foreign organisms into the internal fluids or the colonization of the tunic by encrusting organisms. The most represented chemical classes among the bioactive secondary metabolites isolated from tunicates are alkaloids, polyketides, and peptides [18]. Cytotoxicity against mammalian cell lines and anti-proliferative activity were the most frequently assigned bioactivities. Compounds with cytotoxic and antineoplastic properties isolated from ascidians belong to disparate chemical classes, and three of them have entered clinical trials [20]. Due to their key phylogenetic position in chordate evolution, the ascidians are a powerful model for studying innate immunity [4]. They possess an exclusively innate immune system, including inflammatory, humoral, and cellular responses. On an evolutionary level, inflammation is a highly conserved phenomenon and appears to be an essential first line of defense for both invertebrates and vertebrates. The innate immune system is the major contributor to acute inflammation ^[21][22][21,22], a rapid protective response to microbial infection, tissue injury, and insults [23], and the principal promoter of inflammatory responses often involves infection by microbial invaders or exposure to foreign particles/irritants/pollutants [24].

When host cells capable of innate immune responses encounter pathogenic microbes or other foreign or host irritants, the inflammatory response initiates within minutes. The host cells recognize the stimulus through various sensing mechanisms involving trans-membrane receptors. In Ciona robusta (previously Ciona intestinalis), these interactions transmit signals to the nucleus, resulting in the activation and regulation of numerous genes via both transcriptional and post-transcriptional mechanisms ^{[21][25][26][27][28][29][30][31][32][33]}[21,25,26,27,28,29,30,31,32,33], such as antimicrobial peptides and complement factors ^[34][35][34,35] and proinflammatory cytokines and chemokines which activate endothelial cells and recruit immune system cells to the infection site ^[36][37][38][36,37,38].

The immune system is made up of a complex and dynamic network of cell subsets and mediators that promote host defense from infectious agents or tumor cells and maintain immunological tolerance in the organisms ^[39][40][39,40]. Vertebrate immunity is classically divided into innate and adaptive immune systems that act interdependently based on bidirectional crosstalk [41]. It is well known that the innate immune system provides the first line of defense in immune response and can induce and regulate many different adaptive immunity functions [42].

2. Bioactive Molecules in Ascidians

Bioactive peptides (BPs) are composed of protein fragments or peptides with beneficial metabolic and physiological functions that promote human health; thus, they are excellent molecules for studying human health and disease and potential therapeutics ^[43][44][43,44]. Most have similar structures, such as <20 amino acid residue lengths, and contain hydrophobic residues ^[45][46][45,46]. Based on their modes of action, different classes of BP are evidenced: anticancer (ACP), antiviral, antimicrobial (AMP), anti-oxidative stress, and immunomodulatory peptides ^{[15][16][17][18][19][47][48]}[15,16,17,18,19,47,48]. In the following sections, only AMP and immunomodulatory peptides will be discussed, as they are the only bioactive molecules shown in ascidians.

2.1. Bioactive Molecules with Antimicrobial Activity

AMPs are a class of small peptides that exist widely in nature, and they are an important part of the innate immune system of different organisms. AMPs have a broad range of inhibitory effects against bacteria, fungi, parasites, and viruses. The emergence of antibiotic-resistant microorganisms and increasing concerns about the use of antibiotics have resulted in the development of AMPs, which have good application prospects in medicine, food, animal husbandry, agriculture, and aquaculture. Microorganism resistance to antimicrobials is becoming increasingly severe with the abuse of antibiotics in medicine, agriculture, and animal husbandry. The prevalence of vancomycin-resistant enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) is increasing in clinical medicine, so countermeasures are urgently needed to address these bacterial infections. Research on AMPs is continuously developing, and AMP databases store a considerable amount of data on AMPs. A massive variety of antimicrobials has been extracted from tunicates. They belong to disparate chemical classes, such as polysulfides, alkyl sulfates, terpenes, amino alcohols, spiroketals, alkaloids, furanones, peptides, and others [15]. Some of them are synthesized by symbiotic organisms colonizing the tunic or the internal fluids ^[49][50][49,50]. Most of the known AMPs are produced by ascidian-circulating cells, mainly immunocytes (i.e., cells involved in immune responses) for defense purposes ^{[51][52][53][54][55][56][57][58][59][60]}[51,52,53,54,55,56,57,58,59,60] (Table 1). In Halocynthia roretzi, the tetrapeptides halocyamines A and B are produced by cytotoxic morula cells (MCs) [51], and their cytotoxic activity is likely related to their diphenol rings, representing suitable substrates for the enzyme phenoloxidase (PO), which is also stored inside MCs. The enzyme induces oxidative stress by oxidizing phenols to quinones with the consequent production of ROS [64]. The hemocytes of species H. aurantium synthesize the peptide dicynthaurin and the cationic peptide halocidin [54]. The native peptide of halocidin has a mass of 3443 Da and comprises two different subunits containing 18 amino acid residues and 15 residues which are linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized to make three additional isoforms (15-residue homodimer, 18-residue homodimer, heterodimer). Antimicrobial assays performed with synthetic peptides of halocidin confirmed that congeners of the 18-residue monomer were more active than those of the 15-residue monomer MRSA and multidrug-resistant Pseudomonas aeruginosa. Hemocytes from the solitary tunicate Styela clava contained a family of four α-helical antimicrobial peptides that were purified, sequenced, and named clavanins A, B, C, and D. Clavanins A–D (histidine-rich, -helix peptides) [49] and clavaspirin are synthesized by Styela clava MCs [56]. In lysates of hemocytes of the same species, five cationic antimicrobial peptides, called styelins, were identified and isolated ^[57][58][57,58]. In hemocytes of Styela plicata, the octapeptide plicatamide was isolated [59]. In the tunicates Microcosmus sabatieri and Halocynthia papillosa, antimicrobial activities were detected, and two novel peptides, halocyntin and papillosin, were isolated and characterized. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. A combination of Edman degradation and mass spectrometry obtained a complete peptide characterization. The mature molecules of halocyntin and papillosin comprise 26 and 34 amino acid residues, respectively [60]. The enormous quantity of genomic data has become a promising source of putative AMPs due to progress in bioinformatics ^{[65][66][67][68]}[65,66,67,68]. In C. intestinalis, using genome and expressed sequence tag (EST) data, a putative gene family has been identified exhibiting several structural features typical of AMPs. The synthetic peptide exerted potent antimicrobial activity against various bacteria and against the yeast Candida albicans, but it was not cytolytic for mammalian erythrocytes. Using the synthetic peptide as an antigen, specific antibodies were generated, and the natural parent molecule was localized to a compartment of a distinct hemocyte type, the univacuolar refractile granulocytes [53]. Furthermore, a gene family coding for putative AMPs was identified in the EST database of C. intestinalis and subsequently identified and cloned from the Northern European Ciona subspecies. Molecular analysis revealed that the natural peptide is synthesized and stored in a distinct hemocyte type, the univacuolar non-refractile granulocytes, and that the expression of the gene is markedly upregulated in hemocytes after immune challenge. The peptide proved highly effective against Gram-negative and Gram-positive bacteria, including several human and marine pathogens, as well as the yeast C. albicans. Using two different methods, it was demonstrated that the peptide kills Gram-negative and Gram-positive bacteria by permeabilizing their cytoplasmic membranes. Circular Dichroism (CD) spectroscopy revealed that in the presence of liposomes composed of negatively charged phospholipids, the peptide undergoes a conformational change and adopts an alpha-helical structure. Moreover, the peptide was virtually non-cytolytic for mammalian erythrocytes. Hence, this designed AMP may represent a valuable template for developing novel antibiotics [52]. Ci-MAM-A24, a synthetic AMP derived from a peptide precursor from immune cells of C. intestinalis, is potently active against representatives of Gram-positive and Gram-negative bacteria by permeabilizing their cytoplasmic membrane. The activity of Ci-MAM-A24 against different bacterial pathogens which frequently cause therapeutic problems was tested. Fedders et al. tested the killing capacity of Ci-MAM-A24 against clinically important anaerobic bacteria as well as multiresistant aerobic strains such as MRSA, VRE, extended-spectrum α-lactamase-producers, and multiple-resistant Pseudomonas aeruginosa, and all strains proved to be highly susceptible to Ci-MAM-A24 at low concentrations [69]. Furthermore, an in silico screening method has been developed based on further criteria such as size, amphipathicity, and aggregation propensity, by which 22 potential LCAMP candidates in the Ciona genome were computationally predicted. Among these LCAMP candidates, five novel salt-resistant LCAMPs with broad-spectrum antimicrobial activity were experimentally confirmed. This strategy was also successfully applied to the Xenopus tropicalis genome, suggesting that this method could apply to the in silico screening of any genome [34]. Finally, Lu et al. [61] investigated the potential sORFs encoding AMPs in C. intestinalis, and over 180 peptides deduced from the sORFs were predicted to be AMPs. Among the ten peptides tested, six were found to have significant EST matches, providing strong evidence for gene expression; five were proved to be active against the bacterial strains.

2.2. Bioactive Molecules with Immunomodulatory Effects

A recent idea is to use invertebrates as a source of molecules with potential immunoregulatory activities to improve strategies for modulating human immune system responses ^[65][66][65,66]. The innate immune system is composed of many interdependent cell types and mediators. It is one of the most critical natural systems for protection against many harmful bacteria, viruses, parasites, and fungi in human health, and against autoimmune diseases, cancer, allergies, and infections ^[70][71][70,71]. Preliminary studies have shown evidence supporting a complex interaction between the immune system and tumors [72]. Several innate system immunomodulators have been identified; these include cytokines (interleukins, interferons, and chemokines), substances isolated from microorganisms and fungi (lipopolysaccharides; LPS), and substances isolated from plants (polysaccharides and phenolic compounds) [73]. Tumor cells secrete altered protein products that must be recognized as foreign by the immune effector cells such as B, T, natural killer and natural killer T cells, and type I and II interferons, and perforin which are able to destroy tumor cells ^[74][75][74,75]. Therefore, the enhancement of the host immune response is one of the most important methods for inhibiting tumor growth and maintaining cellular homeostasis without harming the host. The selective modulation of immunity is an emerging concept driven by the tremendous advances in our understanding of this crucial host defense system. Invertebrates have drawn researchers’ interest as potential sources of new bioactive molecules owing to their immunomodulatory activities. An LPS challenge in the ascidian C. intestinalis generates the transcript, Ci8 short, with cis-regulatory elements in the 3′ UTR region which is essential for shaping innate immune responses. The derived amino acidic sequence from in silico analysis showed specific binding to human major histocompatibility complex (MHC) class I and class II alleles. The role of Ci8 short peptide (Table 1) was investigated in a more evolved immune system using human peripheral blood mononuclear cells (PBMCs) as an in vitro model. The biological activities of this molecule include the activation of the 70 kDa TCR ζ chain associated protein kinase (ZAP-70) and T cell receptor (TCR) Vβ oligo clonal selection on CD4+ T lymphocytes as well as increased proliferation and IFN-γ secretion. Furthermore, Ci8 short affects CD4+/CD25high-induced regulatory T cells (iTreg) subset selection, which co-expressed the functional markers TGF-β1/latency-associated protein (LAP) and CD39/CD73 [62]. Furthermore, Colombo et al. [63] evaluated the 3D structure of the C8 short-derived C. robusta chemo-attractive peptide (CrCP) (Table 1) by homology modeling, which showed that CrCP displayed structural characteristics already reported for a short domain of the vertebrate CRK gene, suggesting its possible involvement in cell migration mechanisms. The biological activity of CrCP was studied in vitro using a primary human dermal cell line. In vitro assays demonstrated that CrCP could induce the motility of HuDe cells in both wound healing and chemo-attractive experiments. Furthermore, CrCP modulates the expression of the matrix metalloproteinase-7 (MMP-7) and E-cadherin genes, and it induces the activation of the NF-κB signaling pathway.