Enhanced immunity and hemocytes proliferation by three immunostimulants in tri-spine horseshoe crab Tachypleus tridentatus.

Tachypleus amebocyte lysate (TAL) is crucial in medical testing, but its industry in China has been restricted due to the decline of horseshoe crab population in recent years. Exploring methods of enhancing immunity and rapid hemocytes proliferation is urgent for the industrial horseshoe crab culture. In this study, ß-glucan (G), peptidoglycan (P), and squalene (S) were injected to horseshoe crabs at two concentrations (5 and 10 mg/kg), in order to compare their effects on total hemocyte count (THC), reactive oxygen species (ROS), and non-specific immune enzyme activities. Results showed that the THC, superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased by three immunostimulants at different points of time; ROS was significantly increased except at two squalene groups; lysozyme (LZM) and alkaline phosphatase (AKP) activity were increased except at low dose (5 mg/kg) squalene group; malondialdehyde (MDA) activity was decreased in all treatments; and hemocyanin concentration (HC) changed little during the experiment. At the 48th hour, THC, ROS, SOD, CAT, T-AOC, LZM, and AKP activities were significantly higher in the two peptidoglycan groups than those in the control group; the low dose ß-glucan and squalene groups showed significantly higher SOD and CAT, but their THC and AKP were not significantly different from those of the control group. In general, all three immunostimulants stimulated the hemolymph parameters of horseshoe crabs, notably, peptidoglycan could significantly increase the THC and enzyme activities, suggesting that peptidoglycan can be developed as an efficient immunostimulant for horseshoe crabs.

Methylated glycans as conserved targets of animal and fungal innate defense.

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of ß-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.

Antiviral function of Tachyplesin I against iridovirus and nodavirus.

Antimicrobial peptides (AMPs) are ubiquitously found in living organisms and are an important component in innate immune response. Tachyplesin I is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab, Tachypleus tridentatus. Previous studies have shown that the 17-residue peptide exhibits a wide spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, protozoa, and viruses. However, the efficiencies and defense mechanisms of the Tachyplesin I against fish viruses are still unknown. In this study, Tachyplesin I showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (RGNNV). Synthetic peptides of Tachyplesin I incubated with virus or cells before infection reduced the viral infectivity. Synthetic peptides of Tachyplesin I drastically decreased SGIV and RGNNV titers and viral gene expression. Grouper spleen (GS) and brain (GB) cells over-expressing Tachyplesin I (GS/pcDNA3.1-flag-Tac I and GB/pcDNA3.1-flag-Tac I) support the inhibition of viral infection. Tachyplesin I activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. The promoter activity of IFN-ß and ISRE were significantly up-regulated in cells transfected with pcDNA3.1-flag-Tac I after infection with SGIV and VNNV. These results suggest that Tachyplesin I is importantly involved in host immune responses to invasion of viral pathogens.

Comparative putative metabolites profiling of Tachypleus gigas and Carcinoscorpius rotundicauda hemocytes stimulated with lipopolysaccharide.

Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC-TOF-MS, multivariate analyses, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP > 1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p < 0.05. The metabolites were identified using several databases, and the literature review of the metabolites was reported in the manuscript. Thus, this study has provided further insights into the putative metabolites' presence in the hemocytes of horseshoe crabs that are stimulated and non-stimulated with LPS and their abundance in each species. Several putative metabolites showed they have medicinal values from previous studies.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation.

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Phagocytic activity of Limulus polyphemus amebocytes in vitro.

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.

High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2).

BACKGROUND: Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. RESULTS: In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus), a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella) possess proteins structurally similar to tachylectin-2. CONCLUSIONS: Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1) part of Oculina's innate immunity repertoire, and 2) evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.

Invertebrate recognition protein cross-reacts with an immunoglobulin idiotype.

To estimate the minimal structural requirements for cross-reaction of idiotypic determinants, we determined the capacity of monoclonal antibodies specific for the idiotype of the phosphorylcholine (PC)-binding myeloma protein TEPC-15 for cross-reactivities with the PC-binding, acute-phase protein C-reactive protein (CRP) and the hemagglutinin from the horseshoe crab Limulus polyphemus (limulin), which binds sialic acid and PC. Certain monoclonal antibodies (MAb) to the TEPC-15 idiotype showed strong cross-reactions with CRP and limulin when tested by enzyme-linked immunoadsorbent assays. The specificity of the cross-reactivities was confirmed by testing the binding of the reactive anti-TEPC-15 MAb to both CRP and limulin in the presence of p-nitrophenylphosphorylcholine (pNPPC), N-acetylneuraminic acid, and bovine submaxillary mucin. The binding of the MAb to both CRP and limulin was strongly decreased by pNPPC, partially decreased by free PC, and not affected by N-acetylneuraminic acid or bovine submaxillary mucin. Neither CRP nor limulin showed significant overall sequence homology to vertebrate immunoglobulins. However, CRP, limulin, and TEPC-15 variable region heavy chain (VH) shared short stretches of homology (8-10 amino acids) that mapped to a stretch comprised of the second complementarity determining region and third framework region of the TEPC-15 VH. These results might reflect either evolutionary convergence forced upon molecules of diverse evolutionary histories because of steric requirements of binding the same ligand, or a conservation of primitive combining site gene segments in evolution.

Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation.

The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.

Immunocompetent molecules and their response network in horseshoe crabs.

Horseshoe crab hemocyte selectively responds to bacterial lipopolysaccharides (LPS), which depends critically on the proteolytic activity of the LPS-responsive serine protease zymogen factor C. In response to stimulation by LPS, the hemocyte secretes several kinds of immunocompetent proteins. The coagulation cascade triggered by LPS or beta-1,3-D-glucans (BDG) results in the formation of coagulin fibrils that are subsequently stabilized by transglutaminase (TGase)-dependent cross-linking. Invading pathogens are recognized and agglutinated by lectins and then killed by antimicrobial peptides. Moreover, LPS-triggered hemocyte exocytosis is enhanced by a feedback mechanism in which the antimicrobial peptides serve as endogenous mediators. Factor C also acts as an LPS-sensitive complement C3 convertase. In addition, a sub-cuticular epidermis-derived protein forms a TGase-stabilized mesh at sites of injury. Horseshoe crabs have a sophisticated innate immune response network that coordinately effects pathogen recognition and killing, prophenoloxidase activation, complement activation and TGase-dependent wound healing.

Immune Responses to Gram-Negative Bacteria in Hemolymph of the Chinese Horseshoe Crab, Tachypleus tridentatus.

Chinese horseshoe crab, Tachypleus tridentatus, is an ancient marine arthropod with a long evolutionary history. As a kind of living fossil species, the pathogen defenses of horseshoe crabs entirely depend on the innate immune system. Although, there are abundant immune molecules found in the horseshoe crab hemolymph, the biological mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. In this study, we used high-throughput sequencing at mRNA and protein levels and bioinformatics analysis methods to systematically analyze the innate immune response to Gram-negative bacteria in hemolymph of Chinese horseshoe crab. These results showed that many genes in the complement and coagulation cascades, Toll, NF-κB, C-type lectin receptor, JAK-STAT, and MAPK signaling pathways, and antimicrobial substances were activated at 12 and 24 h post-infection, suggesting that Gram-negative bacteria could activate the hemolymph coagulation cascade and antibacterial substances release via the above pathways. In addition, we conjectured that Toll and NF-κB signaling pathway were most likely to participate in the immune response to Gram-negative bacteria in hemolymph of horseshoe crab through an integral signal cascade. These findings will provide a useful reference for exploring the ancient original innate immune mechanism.

Post-translational protein deimination signatures and extracellular vesicles (EVs) in the Atlantic horseshoe crab (Limulus polyphemus).

The horseshoe crab is a living fossil and a species of marine arthropod with unusual immune system properties which are also exploited commercially. Given its ancient status dating to the Ordovician period (450 million years ago), its standing in phylogeny and unusual immunological characteristics, the horseshoe crab may hold valuable information for comparative immunology studies. Peptidylarginine deiminases (PADs) are calcium dependent enzymes that are phylogenetically conserved and cause protein deimination via conversion of arginine to citrulline. This post-translational modification can lead to structural and functional protein changes contributing to protein moonlighting in health and disease. PAD-mediated regulation of extracellular vesicle (EV) release, a critical component of cellular communication, has furthermore been identified to be a phylogenetically conserved mechanism. PADs, protein deimination and EVs have hitherto not been studied in the horseshoe crab and were assessed in the current study. Horseshoe crab haemolymph serum-EVs were found to be a poly-dispersed population in the 20-400 nm size range, with the majority of EVs falling within 40-123 nm. Key immune proteins were identified to be post-translationally deiminated in horseshoe crab haemolymph serum, providing insights into protein moonlighting function of Limulus and phylogenetically conserved immune proteins. KEGG (Kyoto encyclopaedia of genes and genomes) and GO (gene ontology) enrichment analysis of deiminated proteins identified in Limulus revealed KEGG pathways relating to complement and coagulation pathways, Staphylococcus aureus infection, glycolysis/gluconeogenesis and carbon metabolism, while GO pathways of biological and molecular pathways related to a range of immune and metabolic functions, as well as developmental processes. The characterisation of EVs, and post-translational deimination signatures, revealed here in horseshoe crab, contributes to current understanding of protein moonlighting functions and EV-mediated communication in this ancient arthropod and throughout phylogeny.

Elucidating the function of an ancient NF-kappaB p100 homologue, CrRelish, in antibacterial defense.

The family of NF-kappaB transcription factors essentially regulates immune-related gene expression. Recently, we isolated and characterized the classical NF-kappaB/inhibitor kappaB (IkappaB) homologues from a "living fossil," the horseshoe crab, Carcinoscorpius rotundicauda. Interestingly, this ancient species also harbors another class I NF-kappaB p100 homologue, C. rotundicauda Relish (CrRelish). Similar to Drosophila Relish and the mammalian p100, CrRelish contains both the Rel-homology domains (RHD) and the IkappaB-like domain. In this study, we found that the RHD of CrRelish can recognize horseshoe crab and human kappaB response elements and activate the downstream reporter in vitro, thereby suggesting the evolutionary conservation of this molecule. Pseudomonas aeruginosa infection transcriptionally upregulates CrRelish, which exhibits a dynamic protein profile over the time course of infection. Surprisingly, secondary infection reinduced an upsurge in CrRelish protein expression to a level which overrode the protein degradation at 12 h postinfection. These observations strongly suggest the involvement of CrRelish in antibacterial defense. Secondary infection causes (i) the maintenance of a favorable expression-competent sequence context of the CrRelish gene and/or (ii) the derepression or stabilization of the CrRelish transcript resulting from the primary infection to enable the more rapid expression and accumulation of the CrRelish protein, reflecting apparent signal/immune priming in a repeated infection.

Safety and immunogenicity of high molecular weight polysaccharide vaccine from immunotype 1 Pseudomonas aeruginosa.

The safety and immunogenicity of a high molecular weight polysaccharide from immunotype 1 Pseudomonas aeruginosa were tested in a dose response fashion in adult volunteers. The vaccine lacked toxicity and pyrogenicity for experimental animals. Doses of 50, 75, 150, or 250 microgram were given to groups of individuals as a single dose subcutaneous injection. Doses of 150 and 250 microgram were associated with a significant rise in binding and opsonic antibody at 2 wk postimmunization. Titers remained unchanged for up to 6 mo. The vaccine was almost devoid of toxicity, eliciting no more than a slightly sore and tender arm at the site of injection. High molecular weight polysaccharide antigen appears to induce a good immune response following vaccination that is effective in mediating opsonophagocytic killing of live P. aeruginosa organisms.

The molecular basis of innate immunity in the horseshoe crab.

During the past two decades, the molecular structures and functions have been established for various defense molecules, using horseshoe crab (Limulus) as a model animal. These defense molecules include clotting factors, proteinase inhibitors, lectins, antimicrobial peptides and other humoral factors found mainly in the hemolymph. These components of the cellular and humoral systems, which together comprise innate immunity, defend horseshoe crab effectively from invading microbes.

The role of hemolymph coagulation in innate immunity.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1-->3)-beta-D-glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

Antibiotic peptides as mediators of innate immunity.

Antibiotic peptides are widely distributed in nature. Almost all function as membrane-active agents, disrupting target-cell permeability. Several exhibit a striking selectivity for single-celled microbes over metazoan cells, and as such are amongst the simplest components of the animal's defensive system, which distinguishes environmental microbes from 'self'.

Cloning and characterization of a LPS-regulatory gene having an LPS binding domain in kuruma prawn Marsupenaeus japonicus.

LPS is known as an effective stimulator of the immune system in various animals, including mammals and horseshoe crabs (HSC). Both of these animal groups have suppressive regulatory proteins for the LPS response, e.g. the bactericidal/permeability increasing protein in mammals and anti-LPS factor (ALF) in HSC. Prawns are a valuable aquaculture species, but the regulatory molecules and/or mechanisms that respond to LPS are largely unknown. To investigate the molecular mechanism of the LPS response in kuruma prawns, we cloned a cDNA having a LPS binding domain. A full-length cDNA gene, denoted as M-ALF (Marsupenaeus japonicus ALF-like peptide) was cloned that consisted of 746bp and encoded 123 amino-acid residues. The 3' non-translated region of this gene had the pentamer of ATTTA repeated four times; this is known as sequences for messenger RNA stabilization. Deduced amino-acid sequences showed a 42% homology with Japanese HSC-ALF. In particular, both have clusters of basic and hydrophobic amino acids, indicating that the region is probably binding to lipid A. The mRNA expression was determined for hemocytes, lymphoid organs, hearts, intestines and gills by RT-PCR. The mRNA expression was augmented 1.5-3h after LPS administration in lymphoid organs, but then decreased to normal level at 6h. Synthetic peptides containing Cys30 to Cys51 had LPS neutralizing activity to the Limulus reaction and NO production in RAW264.7 cells. These data suggest that in kuruma prawns, M-ALF acts as a LPS regulator during the acute phase response after invasion of pathogens.

Transglutaminase 2 in inflammation.

Many reports have shown that the expression of transglutaminase 2 (TG 2) is increased in inflammatory diseases. Although during the last several decades multiple physiological roles for TG 2 have been demonstrated in various cell types, its role in the inflammatory process is not yet clear. TG 2 is a crosslinking enzyme that is widely used in many biological systems for tissue stabilization purposes and immediate defense against injury or infection. Aberrant activation of TG 2 activity in tissues contributes to a variety of diseases including neurodegenerative diseases, autoimmune diseases, and cancers. In most cases, TG 2 appears to form an inappropriate protein aggregate that may be cytotoxic enough to trigger inflammation and/or apoptosis. In some cases, such as celiac disease and rheumatoid arthritis, TG 2 is also associated with the pathogenic progression, as well as in the generation of autoantibodies. Recently, we discovered that increased TG 2 activity triggers NF-kappaB activation without I-kappaBalpha kinase signaling. TG 2 induces the polymerization of I-kappaBalpha rather than stimulating I-kappaBalpha kinase. This polymerization of I-kappaB results in the direct activation of NF-kappaB in various cell lines. We also found that TG inhibition reverses NF-kappaB activation. Interestingly, this coincides with the reversal of inflammation in conjunctivitis models by treatment with TG 2 inhibitors. Here, I introduce a new role for TG 2 as a signal modulator, which may suggest a new paradigm for the inflammatory process.

Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity.

In innate immunity, pattern recognition receptors discriminate between self- and infectious non-self-matter. Mammalian homologs of the Drosophila Toll protein, which are collectively referred to as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS) and lipoproteins, whereas the Drosophila Toll protein does not act as a PAMP receptor, but rather binds to Spätzle, an endogenous peptide. In Drosophila, innate immune surveillance is mediated by members of the peptidoglycan recognition protein (PGRP) family, which recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions including the release of antimicrobial peptides and the activation of the prophenoloxidase cascade, the latter effecting localized wound healing, melanization, and microbial phagocytosis. In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte.