Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii)

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12 h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6 h after WSSV challenge, but it was decreased at 2 h and kept the low level to 24 h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2 h and peaked at 12 h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2 h, and decreased at 6 h, and then gradually increased at 12–24 h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria.     

Immune system development during early childhood in tropical Latin America: evidence for the age-dependent down regulation of the innate immune response

The immune response that develops in early childhood underlies the development of inflammatory diseases such as asthma and there are few data from tropical Latin America (LA). This study investigated the effects of age on the development of immunity during the first 5 years of life by comparing innate and adaptive immune responses in Ecuadorian children aged 6–9 months, 22–26 months, and 48–60 months. Percentages of naïve CD4+ T cells declined with age while those of memory CD4⁺ and CD8⁺ T cells increased indicating active development of the immune system throughout the first five years. Young infants had greater innate immune responses to TLR agonists compared to older children while regulatory responses including SEB-induced IL-10 and percentages of FoxP3⁺ T-regulatory cells decreased with age. Enhanced innate immunity in early life may be important for host defense against pathogens but may increase the risk of immunopathology.     

Phagocytosis associated chemiluminescence of hemocytes in Mytilus edulis (Bivalvia)

The respiratory burst associated with phagocytosis by Mytilus edulis hemocytes was investigated by measurement of luminol-dependent chemiluminescence (LDCL). After experimental parameters (number of cells, quantity of stimulus) were determined, the biochemical mechanisms involved in the chemiluminescent process were investigated using inhibitors of oxygen radicals and enzymes. In particular, catechol-like phenols suggested the involvement of NADPH-oxidase and peroxidase in oxidative metabolism of mussel hemocytes. The variability of LDCL response observed among individuals and separated hemocyte subpopulations strongly suggests a variable immunocapacity depending on hemogram composition. Using a specific monoclonal antibody to discriminate different hemocyte types, the eosinophilic granulocytes appeared to exhibit the highest LDCL activity.     

Phox activity of differentiated PLB-985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6-PLA2

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.     

Immunorecognition in the freshwater bivalve, Corbicula fluminea II. Isolation and characterization of a plasma opsonin with hemagglutinating activity

Opsonizing and agglutinating activities of plasma from the freshwater clam, Corbicula fluminea, were found to be inhibited by the sugars, 2-deoxy-D-glucose (deoxy-Glu) and N-acetyl-D-galactosamine (GalNAc). The plasma opsonin/agglutinin was subsequently isolated by a two-step separation procedure. Aldehyde-fixed rabbit erythrocytes (RRBC) were used as a solid-phase plasma opsonin affinity absorbant, and deoxyGlu and GalNAc were used in the eluting buffer to desorb several RRBC-binding plasma proteins. The second step involved the further separation of sugar-eluted proteins by Sephacryl S-200 gel filtration. A plasma protein with an apparent molecular weight of 40 kd on SDS-PAGE under nonreducing conditions was found to possess both agglutinating and opsonizing activities. It was further shown to be composed of two identical 20 kd subunits associated through disulfide linkage(s). Although this protein shares some structural similarity with other bivalve opsonins, differences in native molecular size or subunit structure, agglutinating properties and/or sugar binding specificity support the current hypothesis that naturally occurring plasma opsonins of molluscs represent a heterogeneous group of proteins unified primarily through their lectin-like characteristic of binding specific carbohydrate determinants.     

Characterization and immune function of two intracellular sensors, HmTLR1 and HmNLR, in the injured CNS of an invertebrate

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family.     

The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas)

Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu²⁺. After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation.     

Characterization of a scavenger receptor cysteine-rich-domain-containing protein of the starfish, Asterina pectinifera: ApSRCR1 acts as an opsonin in the larval and adult innate immune systems

Proteins containing a scavenger receptor cysteine-rich (SRCR) domain (SRCR proteins) play an important role in the innate immune system of various metazoan animals. In the starfish Asterina pectinifera, mesenchyme cells and coelomocytes govern the two distinct innate immune systems of the larvae and adults, respectively. Here we identify a cDNA encoding a protein containing nine SRCR domains termed ApSRCR1, and present characterization of the molecular structure, expression, subcellular localization and function of ApSRCR1 protein during ontogenesis of this animal. ApSRCR1 protein is a membrane-type protein with a predicted molecular mass of approximately 120 kDa. During ontogenesis, ApSRCR1 protein is de novo synthesized and localizes to cytoplasmic vesicles in both mesenchyme cells and coelomocytes without translation of maternal mRNA; however, the net production and modification by N-glycosylation of ApSRCR1 protein differs in each cell type. In both types of cell, functional inhibition of ApSRCR1 protein leads to incompetent bacterial clearance and failure of aggregate formation. However, this inhibitory effect is weaker in the mesenchyme cells than in the coelomocytes. In the bacteria-sensitized adult, ApSRCR1 protein is up-regulated and digested to enable its secretion into the coelomic fluid. This secreted form of ApSRCR1 protein can apparently bind to bacteria. Overall, we show that ApSRCR1 protein is finely regulated for expression not only during development but also in a sensitive innate immunological situation, and thereupon acts as an opsonin for bacteria to different extents in the larvae and adults of A. pectinifera.     

Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.     

Identification of Type I IFN in Chinese giant salamander (Andrias davidianus) and the response to an iridovirus infection

The type I IFNs play a major role in the first line of defense against virus infections. In this study, the type I IFN gene designated gsIFN was identified and characterized in the Chinese giant salamander (Andrias davidianus). The genomic DNA of gsIFN contains 5 exons and 4 introns and has a total length of 5622 bp. The full-length cDNA sequence of gsIFN is 1113 bp and encodes a putative protein of 186 amino acids that has a 43% identity to type I IFN of Xenopus tropicalis. The deduced amino acid sequence has the C-terminal CAWE motif, that is mostly conserved in the higher vertebrate type I IFNs. Real-time fluorescence quantitative RT-PCR analysis revealed broad expression of gsIFN in vivo and the highest level expression in blood, kidney and spleen. Additionally, the expression of gsIFN at the mRNA level was significantly induced in peripheral blood leucocytes after stimulation with poly I:C and after infection with the Chinese giant salamander iridovirus (GSIV). A plasmid expressing gsIFN was constructed and transfected into the Chinese giant salamander muscle cell line. Expression of the IFN-inducible gene Mx was up-regulated in the gsIFN-overexpressing cells after GSIV infection. The virus load and titer were significantly reduced compared with that in control cells. Additionally, a lower level of virus major capsid protein synthesis was confirmed by immunofluorescence assay compared to the control cells. These results suggest that the gsIFN gene plays an important role in the antiviral innate immune response.     

Specificity of the innate immune system and diversity of C-type lectin domain (CTLD) proteins in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has become an important model for the study of innate immunity. Its immune system is based on several signaling cascades, including a Toll-like receptor, three mitogen-activated protein kinases (MAPK), one transforming growth factor-beta (TGF-beta), the insulin-like receptor (ILR), and the programmed cell death (PCD) pathway. Furthermore, it also involves C-type lectin domain- (CTLD) containing proteins as well as several classes of antimicrobial effectors such as lysozymes. Almost all components of the nematode immune system have homologs in other organisms, including humans, and are therefore likely of ancient evolutionary origin. At the same time, most of them are part of a general stress response, suggesting that they only provide unspecific defense. In the current article, we re-evaluate this suggestion and explore the level of specificity in C. elegans innate immunity, i.e. the nematode's ability to mount a distinct defense response towards different pathogens. We draw particular attention to the CTLD proteins, which are abundant in the nematode genome (278 genes) and many of which show a pathogen-specific response during infection. Specificity may also be achieved through the differential activation of antimicrobial genes, distinct functions of the immunity signaling cascades as well as signal integration across pathways. Taken together, our evaluation reveals high potential for immune specificity in C. elegans that may enhance the nematode's ability to fight off pathogens.     

Accumulation of rare sex chromosome rearrangements in the African pygmy mouse, Mus (Nannomys) minutoides: a whole-arm reciprocal translocation (WART) involving an X-autosome fusion

Although sex chromosomes are generally the most conserved elements of the mammalian karyotype, those of African pygmy mice show three extraordinary deviations from the norm: (a) asynaptic sex chromosomes, (b) multiple sex–autosome fusions, and (c) modifications of sex determination in some populations/species. In this study we identified, in two sex-reversed females of Mus (Nannomys) minutoides, a fourth rare sex chromosome change: a spontaneous whole-arm reciprocal translocation (WART) between an autosomal Robertsonian pair Rb(13.16) and the sex–autosome fusion Rb(X.1). This represents one of the very few reported cases of WARTs in natura within mammals, and is the first one to involve sex chromosomes. Hence, this finding offers new insights into the mechanisms of chromosomal differentiation in African pygmy mice, as WARTs may have contributed to the extensive diversity not only of autosomal Robertsonian fusions, but also of sex–autosome translocations. More widely, these results provide additional support to previous studies on the house mouse and the common shrew which indirectly inferred the role of WARTs in their karyotypic evolution, and may even help to understand how the fascinating 10 sex chromosome chain of the platypus might have evolved. This accumulation of rare sex chromosome changes in single specimens is, to our knowledge, exceptional among mammals.     

IFN-beta modulates the response to TLR stimulation in human DC: involvement of IFN regulatory factor-1 (IRF-1) in IL-27 gene expression

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.