Functions and therapeutic targets of Siglec-mediated infections, inflammations and cancers.

Siglecs, sialic acid (SA)-binding immunoglobulin (Ig)-like lectins, belong to a family of Ig-like lectins. All Siglecs have at least two domains including an extracellular domain with variable (V) and constant (C)-set immunoglobulin (Ig) regions, and a transmembrane domain. Some of the Siglecs (Siglec-2-12, -17, -E, -F and -G) with three domains including immunoreceptor tyrosine-based inhibitory motif associated with Src homology 2 (SH2) tyrosine phosphatases (SHP1/2) usually deliver an inhibitory signal. Certain Siglecs (Siglec-14, -15, -16 and -H) containing no intracellular domain carry certain basic amino acid in transmembrane domain coupled with immunoreceptor tyrosine-based activating motif for cell activation. The number of Siglec-encoding genes has been correlated to lifespan of mammals, indicating its evolutional advantage on acquisition of Siglecs in humans. Certain polymorphisms of Siglecs have been associated with premature delivery, infection, schizophrenia, allergy, dementia or chronic obstructive pulmonary disease. Siglecs mainly expressing on leukocytes could interact with cis- or trans-SA ligands for cell-cell and host-organism interactions on infections, inflammations and cancers. Amplifying or eliminating the SA-Siglec interactions is a promising strategy to treat cancers, infections and inflammations, based on SA modifications in different linkages or nanoparticle decoration, and on the antibodies in conjugation of chimeric receptor design or toxins.

Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes).

Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN-/- F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN-/- F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.

A member of the immunoglobulin superfamily, orange-spotted grouper novel immune gene EcVig, is induced by immune stimulants and type I interferon.

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response.

Reprint of "review of Dscam-mediated immunity in shrimp and other arthropods".

Although true adaptive immunity is only found in vertebrates, there is increasing evidence that shrimp and other arthropods exhibit immune specificity and immune memory. The invertebrate immune response is now called "innate immunity with specificity" or "immune priming", and its underlying mechanisms are still unclear. However, while vertebrate antibodies have no invertebrate homolog, the Down syndrome cell adhesion molecule (Dscam), which is a hypervariable protein created by alternative splicing, can function as a pathogen-specific recognizing molecule in arthropods. Here we review our current understanding of the Dscam-mediated immune responses in arthropods, especially in shrimp, and show that Dscam may be involved in both general innate immunity and the pathogen-specific immune response.

Review of Dscam-mediated immunity in shrimp and other arthropods.

Although true adaptive immunity is only found in vertebrates, there is increasing evidence that shrimp and other arthropods exhibit immune specificity and immune memory. The invertebrate immune response is now called "innate immunity with specificity" or "immune priming", and its underlying mechanisms are still unclear. However, while vertebrate antibodies have no invertebrate homolog, the Down syndrome cell adhesion molecule (Dscam), which is a hypervariable protein created by alternative splicing, can function as a pathogen-specific recognizing molecule in arthropods. Here we review our current understanding of the Dscam-mediated immune responses in arthropods, especially in shrimp, and show that Dscam may be involved in both general innate immunity and the pathogen-specific immune response.

EcVig, a novel grouper immune-gene associated with antiviral activity against NNV infection.

VHSV-induced genes (VIGs) were first identified in rainbow trout (Oncorhynchus mykiss) and subsequently isolated in a variety of fish. Recent studies have shown that most VIGs have immunological functions against pathogenic infections. However, most research has focused on Vig1, such that our present understanding of these genes in other fish species remains limited. This study isolated a homologue of the uncharacterized O. mykiss Vig-B319 (EcVig) from orange-spotted grouper (Epinephelus coioides). Genomic organization suggests that four EcVig isoforms (EcVig A-D), are generated through alternative splicing. Due to the encoding of 2 immunoglobulin (Ig) domains, the EcVig protein can be considered a member of the immunoglobulin superfamily. The expression of EcVig increased 3 days after hatching (dph) and peaked at 9 dph. This pattern is similar to that displayed by EcMx, an important grouper antiviral gene. Additionally, a tissue tropism assay revealed that EcVig A is the major EcVig isoform present in the tissues considered by this study, with the expression of EcVig A exceeding that of EcVig B. We subsequently investigated whether EcVig expression was induced by the viral pathogen nervous necrosis virus (NNV) or the bacterial pathogen Vibrio anguillarum. Following injection with NNV, the expression levels of EcVig showed significant up-regulation. Conversely, a significant reduction was observed in EcVig expression in brain samples collected from V. anguillarum injected grouper. The overexpression of EcVig A suppressed the replication of NNV in grouper GF-1 cell lines, suggesting that EcVig is an important antiviral factor in the grouper immune responses.

Spawning stress triggers WSSV replication in brooders via the activation of shrimp STAT.

In the early days of shrimp aquaculture, wild-captured brooders usually spawned repeatedly once every 2-4days. However, since the first outbreaks of white spot disease (WSD) nearly 20years ago, captured female brooders often died soon after a single spawning. Although these deaths were clearly attributable to WSD, it has always been unclear how spawning stress could lead to an outbreak of the disease. Using real-time qPCR, we show here that while replication of the white spot syndrome virus (WSSV; the causative agent of WSD) is triggered by spawning, there was no such increase in the levels of another shrimp DNA virus, IHHNV (infectious hypodermal and hematopoietic necrosis virus). We also show that levels of activated STAT are increased in brooders during and after spawning, which is important because shrimp STAT is known to transactivate the expression of the WSSV immediate early gene ie1. Lastly, we used dsRNA silencing experiment to show that both WSSV ie1 gene expression and WSSV genome copy number were reduced significantly after shrimp STAT was knocked-down. This is the first report to demonstrate in vivo that shrimp STAT is important for WSSV replication and that spawning stress increases activated STAT, which in turn triggers WSSV replication in WSSV-infected brooders.

Identification and characterization of DSCAM isoforms isolated from orange-spotted grouper Epinephelus coioides.

The Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin (Ig) superfamily member, was first identified from human and subsequently isolated from both vertebrates and invertebrates. Recent studies have shown that the DSCAM molecule serves diverse functions in neurodevelopment, such as axon guidance and neuronal migration. Most studies on DSCAM, however, have focused on mammals and arthropods, and our present knowledge of bony fish DSCAM is still limited. In this study, orange-spotted grouper Epinephelus coioides was used as an animal model to explore the possible functions of DSCAM. Two DSCAM isoforms were isolated, namely EcDSCAM A and EcDSCAM B, with lengths of 1648 and 2025 amino acids, respectively. The classical domain structure (i.e. 9Ig-4FNIII-1Ig-2FNIII-Transmembrane domain-Cytoplasmic tail) was also found in the coding regions of these two EcDSCAMs. Phylogenetic analysis showed that in the vertebrate DSCAM clade, the EcDSCAMs and various teleost DSCAMs were clustered into a subclade. Real-time PCR revealed that EcDSCAM B is the major EcDSCAM isoform, with the expression of EcDSCAM B being significantly higher than that of EcDSCAM A. During the first 14days after hatching (dph), increases in the expression of the two EcDSCAMs were observed at 2-4 and 8-11dph. EcDSCAM is expressed mainly in the intestine, nerve-related tissues, and stomach. Optic nerve transection analysis showed that EcDSCAM was up-regulated during optic nerve regeneration after optic nerve injury. We also investigated whether DSCAM expression was affected by viral nervous necrosis (VNN) disease or vibriosis. We found that when grouper were challenged with nervous necrosis virus (NNV), there were no meaningful changes in DSCAM expression, but challenge with Vibrio anguillarum led to a decrease in EcDSCAM levels in the brain. This decrease may be related to the pathogenesis of V. anguillarum.