Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity.

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota.

Mammalian gut microbiota are integral to host health. However, how this association began remains unclear. We show that in basal chordates the gut space is radially compartmentalized into a luminal part where food microbes pass and an almost axenic peripheral part, defined by membranous delamination of the gut epithelium. While this membrane, framed with chitin nanofibers, structurally resembles invertebrate peritrophic membranes, proteome supports its affinity to mammalian mucus layers, where gut microbiota colonize. In ray-finned fish, intestines harbor indigenous microbes, but chitinous membranes segregate these luminal microbes from the surrounding mucus layer. These data suggest that chitin-based barrier immunity is an ancient system, the loss of which, at least in mammals, provided mucus layers as a novel niche for microbial colonization. These findings provide a missing link for intestinal immune systems in animals, revealing disparate mucosal environment in model organisms and highlighting the loss of a proven system as innovation.

Analysis of the expression patterns of the cytokine receptor family B (CRFB) and interferon gamma receptor (IFNGR) in Dabry's sturgeon (Acipenser dabryanus).

Teleost fish have more complex interferon receptor systems than mammals. In the present study, genes encoding four cytokine receptor family B (CRFBs) and two interferon gamma receptors (IFNGRs) in Dabry's sturgeon (Acipenser dabryanus) were identified by RNA-sequencing. Sequence analysis revealed that the Dabry's sturgeon CRFBs and IFNGRs contained several conserved characteristics features, including signal peptides and a transmembrane domain. Phylogenetic analysis suggested that they belong to the CRFB3, CRFB5, and IFNGR protein families, and were named CRFB3a, CRFB3b, CRFB5a, CRFB5b, IFNGR1, and IFNGR2. The expression patterns of the CRFB and IFNGR genes were investigated in Dabry's sturgeon. The expression levels of CRFB5a, CRFB5b, and IFNGR1 showed no significant changes, suggesting that those genes do not mediate embryonic development. By contrast, the high expression levels of CRFB3a, CRFB3b, and IFNGR2 in the fertilized egg, 16-cell phase, and initial blastula stage implied the existence of maternally expression in the oocyte and association with embryonic development. Tissue distribution analysis revealed that the CRFB and IFNGR proteins have potential functions in immune and non-immune tissue compartments. Comprehensive analysis in Dabry's sturgeon revealed that the expression fold changes of CRFB3a, CRFB3b, CRFB5a, and CRFB5b in Dabry's sturgeon stimulated with poly I:C were higher than those in fish administrated with lipopolysaccharide (LPS). Conversely, the fold changes IFNGRs mRNA levels stimulated with LPS were higher than those in fish administrated with poly I: C. CRFB5a and IFNGR2 genes showed the earliest responses to the poly I: C, and the CRFB5a and IFNGR1 genes showed the earliest responses to LPS. These results implied that CRFB5a has important role in the IFN immune response. Our findings indicated that the Dabry's sturgeon CRFB and IFNGR genes have important functions in antiviral and antibacterial immune responses. The differential responses of these genes to poly I: C and LPS implied differences in the defense mechanisms against viruses and bacteria.

Immune-directed support of rich microbial communities in the gut has ancient roots.

The animal gut serves as a primary location for the complex host-microbe interplay that is essential for homeostasis and may also reflect the types of ancient selective pressures that spawned the emergence of immunity in metazoans. In this review, we present a phylogenetic survey of gut host-microbe interactions and suggest that host defense systems arose not only to protect tissue directly from pathogenic attack but also to actively support growth of specific communities of mutualists. This functional dichotomy resulted in the evolution of immune systems much more tuned for harmonious existence with microbes than previously thought, existing as dynamic but primarily cooperative entities in the present day. We further present the protochordate Ciona intestinalis as a promising model for studying gut host-bacterial dialogue. The taxonomic position, gut physiology and experimental tractability of Ciona offer unique advantages in dissecting host-microbe interplay and can complement studies in other model systems.

RNA viruses and the host microRNA machinery.

Gene silencing by small RNAs (sRNAs) occurs in all three domains of life. In recent years, our appreciation of the diverse functions of sRNAs has increased, and we have identified roles for these RNAs in cellular differentiation, fitness and pathogen defence. Interestingly, although plants, nematodes and arthropods use sRNAs to combat viral infections, chordates have replaced this defence strategy with one based exclusively on proteins. This limits chordate use of sRNAs to the silencing of genome-encoded transcripts and has resulted in viruses that do not perturb sRNA-related cellular processes. This evolutionary phenomenon provides an opportunity to exploit the pre-existing chordate sRNA pathways in order to generate a range of virus-based biological tools. Here, I discuss the relationship between sRNAs and RNA viruses, detail how microRNA expression can be harnessed to control RNA viruses and describe how RNA viruses can be designed to deliver sRNAs.

Particularity and universality of a putative Gram-negative bacteria-binding protein (GNBP) gene from amphioxus (Branchiostoma belcheri): insights into the function and evolution of GNBP.

Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. The roles of GNBP in innate immunity of arthropods and molluscs have recently been reported. However, the GNBP gene has not been characterized in the species of higher evolutionary status yet. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). First, we identified and cloned the AmphiGNBP and found that the AmphiGNBP encodes a putative protein with 558 amino acids, which contains a conserved ß-1, 3-glucan recognizing and binding domain. Second, we found that the AmphiGNBP encodes two extra WSC (cell Wall integrity and Stress response Component) domains, which are unique in AmphiGNBP protein. The two WSC domains of AmphiGNBP protein coupled with the expansion of amphioxus immunity repertoire might undergo intensive domain shuffling during the age of the Cambrian explosion. Finally, we found that the AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine, and the expression of AmphiGNBP was affected after LPS stimulation. In conclusion, our findings disclose the particularity and universality of AmphiGNBP and provide profound insights into the function and evolution of GNBP.

Identification and characterization of a putative lipopolysaccharide-induced TNF-α factor (LITAF) gene from Amphioxus (Branchiostoma belcheri): an insight into the innate immunity of Amphioxus and the evolution of LITAF.

Innate immunity defenses against infectious agent in all multicultural organisms. TNF-α is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-α factor (LITAF) functions as a transcription factor for regulating the expression of TNF-α as well as various inflammatory cytokines in response to LPS stimulation. The physiological significance of LITAF gene in the innate immunity of various animals has recently been reported. However, no LITAF gene has yet been identified in amphioxus, which is the best available stand-in for the proximate invertebrate ancestor of the vertebrates. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with ~1.6 kb in length has a 827bp cDNA transcription product which encodes a putative protein with 127 amino acids containing conserved LITAF-domain, and the deduced amino acid of AmphiLITAF shared 37-60% similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family.

The amphioxus genome provides unique insight into the evolution of immunity.

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.

Evolution of the IL17 receptor family in chordates: a new subfamily IL17REL.

The human interleukin 17 receptor (IL17R) family plays a critical role in inflammatory responses and contributes to the pathology of many autoimmune diseases. So far, five members, IL17RA to IL17RE, have been identified. Recently, some IL17R genes have been identified in non-mammalian species, such as zebrafish IL17RD; however, there are no reports on the evolutionary history of this complex gene family through comparative phylogenetic approaches. Here, we concentrated on the IL17R evolution in chordates. There are two IL17Rs in the genome of the basal chordate amphioxus: IL17RA and IL17RD. After two rounds of whole genome duplications, these two IL17R genes expanded into five early vertebrate IL17R genes, IL17RA to IL17RE. IL17RA and IL17RD are found in most vertebrates, whereas the other three, IL17RB, ILR17RC, and IL17RE, underwent some loss in vertebrates during evolution. Our sequence and structure analyses reveal functional similarities and distinctions between the different IL17Rs. Based on similarity searches for IL17R-like proteins within chordate sequences, a group of IL17RE-like (IL17REL) proteins were identified from mammalians to lower vertebrates. In silico and expression analyses on the novel IL17RELs showed that this group of receptors is highly conserved across species, indicating that IL17REL may represent a unique subfamily of IL17Rs.

An ancient molecule with novel function: Alanine aminotransferase as a lipopolysaccharide binding protein with bacteriocidal activity.

Alanine aminotransaminase (ALT) has been identified from bacteria to plants to animals including humans. The increase in serum ALT is regarded as an index for clinical diagnosis of liver function in humans. However, ALT elevation is also reported in non-liver injury conditions and in apparently healthy people, suggesting it may play a fundamental role physiologically. Herein we isolated an alt homolog, Amphialt, from Branchiostoma japonicus, an intermediatary species from invertebrates to vertebrates, which encoded a polypeptide of 500 amino acids with more than 62 and 52% sequence identity to vertebrate and invertebrate ALT isoenzymes, respectively. It was constitutively expressed in many tissues including the hepatic caecum, the precursor of liver, and its expression in the caecum was significantly up-regulated by challenge with lipopolysaccharides (LPS). Strikingly, recombinant AmphiALT, with a specific activity of 0.114±0.02U/mg, was capable of specifically binding to the Gram-negative bacteria Escherichia coli and Aeromonas hydrophila and to their conserved molecule LPS, as well as inhibiting the growth of E. coli and causing its lysis. In contrast, AmphiALT did not bind to the Gram-positive bacteria Staphyloccocus aureus and Bacillus subtilis as well as their conserved molecule LTA. In addition, a high homology noted between amphioxus and mammalian ALT sequences suggested a functional conservation of ALT evolutionarily, hinting at the clue that mammalian ALT may also play an antibacterial role similar to that of AmphiALT. Taken together, it is proposed that AmphiALT is an immune-relevant molecule capable of identifying LPS and causing damage to Gram-negative bacteria like E. coli and A. hydrophila. It also bolsters the notion that the hepatic caecum of amphioxus is the precursor of vertebrate liver, acting as a major tissue in acute phase response.

Amphioxus SARM involved in neural development may function as a suppressor of TLR signaling.

Among five Toll/IL-1R resistance adaptors, sterile alpha and Toll/IL-1R resistance motif containing protein (SARM) is the only one conserved from Caenorhabditis elegans to human. However, its physiologic roles are hardly understood, and its involvement in TLR signaling remains debatable. In this study, we first demonstrated a predominant expression of amphioxus SARM (Branchiostoma belcheri tsingtauense SARM) in neural cells during embryogenesis and its predominant expression in the digestive system from larva to adult, suggesting its primitive role in neural development and a potential physiologic role in immunity. We further found that B. belcheri tsingtauense SARM was localized in mitochondria and could attenuate the TLR signaling via interacting with amphioxus MyD88 and tumor necrosis receptor associated factor 6. Thus, amphioxus SARM appears unique in that it may play dual functions in neural development and innate immunity by targeting amphioxus TLR signaling.

Immune parameters in the humoral fluids of amphioxus Branchiostoma belcheri challenged with Vibrio alginolyticus.

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including lysozyme, antimicrobial activity, microbial agglutinin and haemagglutinin in amphioxus humoral fluids before and after Vibrio alginolyticus challenge. After challenged with V. alginolyticus, the lysozyme activity, growth inhibiting activities against Escherichia coli and V. alginolyticus and microbial agglutinating activities against Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus were all increased significantly and haemagglutinating activities against rabbit and human A and O erythrocytes in the humoral fluids were all increased earlier. In contrast, the agglutinating activities against Vibrio harvey and E. coli in the humoral fluids were reduced in response to V. alginolyticus challenge and the haemagglutinating activity against human B erythrocytes increased later.

Ovochymase in amphioxus Branchiostoma belcheri is an ovary-specific trypsin-like serine protease with an antibacterial activity.

Ovochymases have been shown to be present in vertebrates; little information is available at present regarding ovochymase in invertebrates. Here we isolated a cDNA encoding an ovochymase homolog from amphioxus Branchiostoma belcheri, named BbOvc. The cDNA contained a 1248bp open reading frame corresponding to a deduced protein of 415 amino acids with a predicted molecular mass of approximately 44.4kDa. Phylogenetic analysis showed that BbOvc was located at the base of its vertebrate counterparts, suggesting that it represents the archetype of vertebrate ovochymases. BbOvc is found to display a tissue- and stage-specific expression pattern, with a predominant expression in the ovary of sexually matured females and in the early stage embryos (1-16-cell embryos). The recombinant ovochymase expressed in vitro shows a trypsin-like activity capable of hydrolysing the trypsin prototypic substrate N(a)-benzoyl-l-arginine ethyl ester (60UBAEE/mg), which can be inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. It also exhibits an antibacterial activity capable of inhibiting the growth of bacteria like E. coli and V. parahaemolyticus. Taken together, these data indicate that BbOvc is a novel ovochymase with an antibacterial activity and offer first clues to its role as an immune-relevant molecule which may protect the early embryos from pathogenic attacks.

Identification, expression and antibacterial activity of a tachylectin-related homolog in amphioxus Branchiostoma belcheri with implications for involvement of the digestive system in acute phase response.

Tachylectin-related proteins have been identified in various organisms from slime molds to sponges to bony fish, yet little is known to date about it in protochordate amphioxus, an important organism occupying a nodal position from invertebrates to vertebrates. Moreover, if the protein acts as an immune-relevant molecule remains controversial. Here we demonstrated the presence of a tachylectin-related gene in Branchiostoma belcheri. The predicted gene product, termed BbTL, consists of 305 amino acids with a putative N-terminal signal peptide and 6 tachylectin-typical tandem repeats of 30-33 amino acids. In situ hybridization histochemistry indicates a tissue-specific expression pattern of BbTL in adult amphioxus with the most abundant expression in the hepatic caecum and hind-gut. Quantitative real-time PCR reveals that challenge with LPS results in a significant up-regulation of BbTL expression in the guts. In addition, the recombinant BbTL expressed in Pichia pastoris is able to inhibit the growth of Gram-negative bacterium Escherichia coli in a dose-dependent manner. All these suggest that BbTL, like most other tachylectin-related proteins, is involved in the host immune defense, and the digestive system of B. belcheri appears the major immune tissue responding to LPS challenge.

Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.

Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus (Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen beta and gamma chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.

A short-form C-type lectin from amphioxus acts as a direct microbial killing protein via interaction with peptidoglycan and glucan.

To investigate the evolution and immune function of C-type lectin in amphioxus, the primitive representative of the chordate phylum, we identified three C-type lectins consisting solely of a carbohydrate recognition domain and N-terminal signal peptide and found that they had distinct express patterns in special tissues and immune response to stimulations analyzed by quantitative real-time PCR. We characterized the biochemical and biological properties of AmphiCTL1, which was dramatically up-regulated in amphioxus challenged with Staphylococcus aureus, Saccharomyces cerevisiae, and zymosan. Immunohistochemistry demonstrated that the localization of AmphiCTL1 protein was exclusively detected in the inner folding tissues of the hepatic diverticulum. Recombinant AmphiCTL1 was characterized as a typical Ca2+-dependent carbohydrate-binding protein possessing hemagglutinating activity, preferentially bound to all examined four Gram-positive bacteria and two yeast strains, but had little binding activity toward four Gram-negative bacteria we tested. It aggregated S. aureus and S. cerevisiae in a Ca2+-dependent manner and specifically bound to insoluble peptidoglycan and glucan, but not to LPS, lipoteichoic acid, and mannan. Calcium increased the intensity of the interaction between AmphiCTL1 and those components, but was not essential. This lectin directly killed S. aureus and S. cerevisiae in a Ca2+-independent fashion, and its binding to microorganism cell wall polysaccharides such as peptidoglycan and glucan preceded microbial killing activity. These findings suggested that AmphiCTL1 acted as a direct microbial killing C-type lectin through binding microbial targets via interaction with peptidoglycan and glucan. Thus, AmphiCTL1 may be an evolutionarily primitive form of antimicrobial protein involved in lectin-mediated innate immunity.

Proteomic approach for caudal trauma-induced acute phase proteins reveals that creatine kinase is a key acute phase protein in amphioxus humoral fluid.

Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid accompanied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned.

Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in amphioxus Branchiostoma belcheri with implications for GILT in innate immune response.

An amphioxus cDNA, AmphiGILT, encoding GILT protein was isolated from the gut cDNA library of Branchiostoma belcheri. It codes for a deduced protein of 254 amino acids, which has all the main features typical of GILT proteins including the signature sequence CQHGX(2)CX(2)NX(4)C, CXXC motif and 11 conserved cysteines. Phylogenetic analysis showed that AmphiGILT and sea urchin GILT clubbed together and positioned at the base of vertebrate GILT clade, suggesting that both AmphiGILT and sea urchin GILT might share some characteristics of the archetype of vertebrate GILT proteins. The genomic DNA sequence of B. floridae contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. AmphiGILT was expressed in a tissue-specific manner with the most abundant mRNA in the digestive system including hepatic caecum and hind-gut. It was also found that mammalian IFN-gamma only exerted a slight effect on the expression of GILT gene in amphioxus, forming a contrast to the marked induction of human and mouse GILT expression by IFN-gamma. Taken the absence of the adaptive immune system including MHC class II molecules and lymphocytes into consideration, these results suggest that AmphiGILT is highly likely to play a role in the innate immune responses in amphioxus.