Identification and immunoregulatory function of neuromedin U (Nmu) in the Japanese pufferfish Takifugu rubripes

In this study, immunoregulatory function of neuromedin U (Nmu) in the teleost fish Fugu (Takifugu rubripes) was characterized. Three splicing variants of nmu mRNA encoding preproNMUs consisting of 164 (Nmu1), 139 (Nmu2), and 129 (Nmu3) amino acid residues were found in Fugu.The biologically active C-terminal region of Fugu Nmu showed high homology among fish and other vertebrate NMUs. The genomic organization of Fugu nmu differed from those of zebrafish and mammals. However, in phylogenetic analysis, Fugu Nmu formed a cluster with NMUs of other vertebrates, in addition to neuromedin S. The splicing variants of mRNA were identified in various tissues. Nmu-21 and Nmu-9 were purified as endogenous peptides from Fugu intestine. The synthetic Nmu-21 peptide activated phagocytic cells, and elevated the expression of cytokine mRNA in peripheral blood leukocytes.     

Identification,origin and evidence for retained functionality of two IκBα paralogs in Megalobrama amblycephala

IκBα plays an essential role in the innate immune response in mammals. We found two functional IκBα paralogs, originating from the teleost-specific genome duplication, in Megalobrama amblycephala: maIκBαa and maIκBαb. Their size (936/933 bp) and structure are highly analogous to known orthologs. mRNA expression was analysed by qPCR in spleen, liver, kidney, intestine and gills. Apart from maIκBαb in gills (<0.001-fold), both paralogs were constitutively expressed in all tissues. Differential expression was observed in gills (high for maIκBαa) and liver: maIκBαa - 2nd lowest (0.47), maIκBαb - 2nd highest (4.25). Both paralogs (mRNA) were upregulated in liver, spleen and kidney after a bacterial (Aeromonas hydrophila) challenge. In spleen, expressions peaked at 12 h post injection (hpi) (maIκBαa = 14.3-fold, maIκBαb = 21.3-fold), but only maIκBαb was highly upregulated at 4, 24 and 120 hpi. In liver, both were upregulated early, but maIκBαa peaked at 4 hpi (15.2-fold) and maIκBαb at 12 hpi (9.8-fold). In kidney, maIκBαa was highly upregulated only at 12 hpi (8.7-fold), and maIκBαb at 4 (peak - 8.2-fold), 12 and 24 hpi. The results indicate that both IκBα paralogs have retained their functionality, that they are structurally and functionally homologous to IκBα orthologs described in other animal species, and that they both play an important role in the innate immune system of M. amblycephala.     

New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes

Neuropeptide Y (NPY) plays different roles in mammals such as: regulate food intake, memory retention, cardiovascular functions, and anxiety. It has also been shown in the modulation of chemotaxis, T lymphocyte differentiation, and leukocyte migration. In fish, NPY expression and functions have been studied but its immunomodulatory role remains undescribed. This study confirmed the expression and synthesis of NPY in S. salar under inflammation, and validated a commercial antibody for NPY detection in teleost. Additionally, immunomodulatory effects of NPY were assayed in vitro and in vivo. Phagocytosis and superoxide anion production in leukocytes and SHK cells were induced under stimulation with a synthetic peptide. IL-8 mRNA was selectively and strongly induced in the spleen, head kidney, and isolated cells, after in vivo challenge with NPY. All together suggest that NPY is expressed in immune tissues and modulates the immune response in teleost fish.     

A new subfamily of penaeidin with an additional serine-rich region from kuruma shrimp (Marsupenaeus japonicus) contributes to antimicrobial and phagocytic activities

Penaeidins are an important family of antimicrobial peptides (AMPs) in penaeid shrimp. To date, five groups of penaeidins have been identified in penaeid shrimp. All are composed of a proline-rich N-terminus and a C-terminus containing six cysteine residues engaged in three disulfide bridges. In this study, a new type of penaeidin from Marsupenaeus japonicus was identified. The full-length penaeidin contains a unique serine-rich region and a penaeidin domain, which consists of a proline-rich region and a cysteine-rich region. Here, we classify all penaeidins into two subfamilies. All reported penaeidins are in subfamily I, and the new penaeidin identified in M. japonicus is designated as Penaeidin subfamily II (MjPen-II). MjPen-II was expressed in hemocytes, heart, hepatopancreas, gills, stomach and intestine, and was upregulated after bacterial challenge. A liquid bacteriostatic assay showed that MjPen-II had antibacterial activity to some Gram-positive and Gram-negative bacteria. MjPen-II could bind to bacteria by binding to polysaccharides on the surface of bacteria, thus promoting bacterial agglutination. The serine-rich region enhanced the agglutination activity of MjPen-II. The proline-rich domain had a stronger bacterial-binding activity and polysaccharide-binding activity than the cysteine-rich domain. MjPen-II was also found to be involved in the phagocytosis of bacteria and efficiently improved the phagocytosis rate. Therefore, MjPen-II eliminates bacteria through direct bacterial inhibition as well as by promoting phagocytosis in shrimp.     

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides

Celiac disease (CD) is a common CD4⁺ T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo.We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5⁺ CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye.Avenin-specific responses were observed in 6/73 HLA-DQ2.5⁺ CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability.Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.     

Differential expression of microRNAs in shrimp Marsupenaeus japonicus in response to Vibrio alginolyticus infection

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including mammals, plants, insects, nematodes and viruses. They are known to have antiviral functions in crustaceans such as shrimp Marsupenaeus japonicas. However, little is known about the role of miRNAs against bacterial infection in this shrimp caused by Vibrio alginolyticus. We performed small RNA sequencing to characterize the differentially expressed microRNAs in V. alginolyticus challenged shrimp, in comparison to that in control uninfected shrimp, at 24 h and 48 h. In total, 55 host miRNAs were differentially expressed in response to the infection and most of these were downregulated at both the time-points. TargetScan and miRanda algorithms showed that the target genes of these down-regulated miRNAs were related to innate immune functions such as production of phenoloxidase enzyme, apoptosis and phagocytosis. Further, gene ontology analysis revealed that many immune signaling pathways were mediated by these miRNAs. This study is one of the earliest attempts at characterizing shrimp miRNAs that respond to V. alginolyticus infection, and will help unravel the miRNA pathways involved in antibacterial action in shrimp.     

Late-onset myasthenia gravis – CTLA4low genotype association and low-for-age thymic output of naïve T cells

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG – its associations with the CTLA4^{high/gain-of-function} +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4^low +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of ‘true LOMG’ and an LOMG/early-onset MG overlapping group of patients between 40 and 60.     

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3

Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense.     

Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (LIF)

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system (CNS). Many nerve axons are insulated by a myelin sheath and their demyelination not only prevents saltatory electrical signal conduction along the axons but also removes their metabolic support leading to irreversible neurodegeneration, which currently is untreatable. There is much interest in potential therapeutics that promote remyelination and here we explore use of leukaemia inhibitory factor (LIF), a cytokine known to play a key regulatory role in self-tolerant immunity and recently identified as a pro-myelination factor. In this study, we tested a nanoparticle-based strategy for targeted delivery of LIF to oligodendrocyte precursor cells (OPC) to promote their differentiation into mature oligodendrocytes able to repair myelin. Poly(lactic-co-glycolic acid)-based nanoparticles of ∼120 nm diameter were constructed with LIF as cargo (LIF-NP) with surface antibodies against NG-2 chondroitin sulfate proteoglycan, expressed on OPC. In vitro, NG2-targeted LIF-NP bound to OPCs, activated pSTAT-3 signalling and induced OPC differentiation into mature oligodendrocytes. In vivo, using a model of focal CNS demyelination, we show that NG2-targeted LIF-NP increased myelin repair, both at the level of increased number of myelinated axons, and increased thickness of myelin per axon. Potency was high: a single NP dose delivering picomolar quantities of LIF is sufficient to increase remyelination.Impact statementNanotherapy-based delivery of leukaemia inhibitory factor (LIF) directly to OPCs proved to be highly potent in promoting myelin repair in vivo: this delivery strategy introduces a novel approach to delivering drugs or biologics targeted to myelin repair in diseases such as MS.     

Identification of in-vivo induced genes of Streptococcus suis serotype 2 specially expressed in infected human

Streptococcus suis (S. suis) serotype 2 usually cause infection in swine. Recently, two large-scale outbreaks in China with severe streptococcal toxic shock syndrome (STSS) and high mortality raised worldwide concern to human S. suis infection. To reveal the molecular pathogenesis of S. suis 2 during human infection, in-vivo induced antigen technology (IVIAT) was applied to identify the in-vivo induced genes (ivi genes) of S. suis 05ZYH33. The ivi genes are specifically expressed or up-regulated in-vivo and always associated with the in-vivo survival and pathogenicity of pathogens. In present study, convalescent sera from S. suis 05ZYH33 infected patients were pooled and fully adsorbed with in-vitro grown S. suis 05ZYH33 and Escherichia coli BL21 (DE3). Genomic expression library of 05ZYH33 was repeatedly screened with colony immunoblot assay using adsorbed sera. Finally, 19 genes were assessed as ivi genes of 05ZYH33. Fifteen of 19 genes encode proteins with biological functions in substance transport and metabolism, cell structure biogenesis, cell cycle control, replication, translation and other functions. The 4 remaining genes encode proteins with unknown functions. Of the 19 ivi genes, five (SSU05_0247, 0437, 1577, 1664 and 2144) encode proteins with no immunoreactivity to control sera from healthy individuals never exposed to 05ZYH33. The successful identification of ivi genes not only sheds light on understanding the pathogenesis of S. suis 05ZYH33 during its human infection, but also provides potential targets for the developments of new vaccines, therapeutic drugs and diagnostic reagents against human S. suis infection.     

Effect of two monoterpene phenols on antioxidant defense system in Candida albicans

Thymol and carvacrol from the class of monoterpene phenols are one of the most potent plant essential oil components possessing antimicrobial effects. Known for their wide bioactive spectrum, these positional isomers of isopropyl cresol deplete ergosterol content, compromise membrane permeability, block efflux pumps and restore antifungal susceptibility to fluconazole in resistant Candida strains. Exposure to these natural compounds induces a cascade of stress responses, which are important to comprehend their microbicidal mechanisms. This study evaluates the antioxidant defense response to lower concentrations of thymol and carvacrol in Candida albicans. The antioxidant defense responses in C. albicans are important for developmental mechanisms pertaining to resistance against the immune system, infection establishment and drug resistance. In this view, primary and secondary antioxidant defense enzymes, and oxidative stress markers including glutathione and lipid peroxidation were determined in C. albicans cells exposed to lower concentrations of thymol and carvacrol. These compounds were found to induce oxidative stress and compromised the antioxidant defense system in C. albicans at lower concentrations. This study helps in understanding the ‘in cell’ antifungal mechanisms of natural monoterpene phenols originating from oxidative stress. Thymol and carvacrol induced membrane deterioration reported earlier, is further explained as a result of a toxic radical cascade mediated by lipid peroxidation. Findings reinforce the observed toxic oxidizing effects of these compounds as a consequence of direct damage to antioxidant components and not to their genetic manipulations.     

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.     

MHC in a monogamous lizard – Characterization of class I MHC genes in the Australian skink Tiliqua rugosa

The major histocompatibility complex (MHC) is a highly variable region of vertebrate genomes that encodes cellular proteins involved in the immune response. In addition to the benefits of MHC research in understanding the genetic basis of host resistance to disease, the MHC is an ideal candidate for studying genetic diversity under strong natural selection. However, the MHC of many non-model vertebrate taxa are poorly characterized, hindering an understanding of disease resistance and its application to conservation genetics in these groups. Squamates (lizards and snakes) remain particularly underrepresented despite their being the most diverse order of non-avian sauropsids. We characterized MHC class I sequence diversity from an Australian skink, the sleepy lizard (Tiliqua rugosa), using both cDNA and genomic sequence data and also present genomic class I sequences from the related skinks Tiliqua adelaidensis and Egernia stokesii. Phylogenetic analysis of Tiliqua and other published sqamate MHC class I sequences suggest that MHC diverged very early in Tiliqua compared with the other studied squamates. We identified at least 4 classical MHC class I loci in T. rugosa and also shared polymorphism among T. rugosa, T. adelaidensis and E. stokesii in the sequences encoding peptide-binding α1 and α2 domains.     

Tolerogenic dendritic cells specific for β2-glycoprotein-I Domain-I,attenuate experimental antiphospholipid syndrome

Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (β₂GPI) tDCs or β₂GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with β₂GPI or D-I or D-V derivatives. Our results revealed that β₂GPI related tDCs phenotype includes CD80^high, CD86^high CD40^high MHC class II^high. The miRNA profiling encompass miRNA 23b^high, miRNA 142-3p^low and miRNA 221^low. In addition the β₂GPI related tDCs showed reduced secretion of IL-1β, IL-12 and IL-23. D-I tDCs treatment was more efficient than β₂GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- β₂GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to β₂GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFβ. Tolerance was successfully transferred by Treg cells from the tolerized mice to β₂GPI immunized mice. We conclude that predominantly D-I-tDCs and β₂GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- β₂GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that β2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.     

Plasmodium and mononuclear phagocytes

Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host.     

Occurrence,integrity and functionality of AcaML1–like viruses infecting extreme acidophiles of the Acidithiobacillus species complex

General knowledge on the diversity and biology of microbial viruses infecting bacterial hosts from extreme acidic environments lags behind most other econiches. In this study, we analyse the AcaML1 virus occurrence in the taxon, its genetic composition and infective behaviour under standard acidic and SOS-inducing conditions to assess its integrity and functionality. Occurrence analysis in sequenced acidithiobacilli showed that AcaML1-like proviruses are confined to the mesothermophiles Acidithiobacillus caldus and Thermithiobacillus tepidarius. Among A. caldus strains and isolates this provirus had a modest prevalence (30%). Comparative genomic analysis revealed a significant conservation with the T. tepidarius AcaML1-like provirus, excepting the tail genes, and a high conservation of the virus across strains of the A. caldus species. Such conservation extends from the modules architecture to the gene level, suggesting that organization and composition of these viruses are preserved for functional reasons. Accordingly, the AcaML1 proviruses were demonstrated to excise from their host genomes under DNA-damaging conditions triggering the SOS-response and to produce DNA-containing VLPs. Despite this fact, under the conditions evaluated (acidic) the VLPs obtained from A. caldus ATCC 51756 could not produce productive infections of a candidate sensitive strain (#6) nor trigger it lysis.     

Distinct functional responses to stressors of bone marrow derived dendritic cells from diverse inbred chicken lines

Differences in responses of chicken bone marrow derived dendritic cells (BMDC) to in vitro treatment with lipopolysaccharide (LPS), heat, and LPS + heat were identified. The Fayoumi is more disease resistant and heat tolerant than the Leghorn line. Nitric Oxide (NO) production, phagocytic ability, MHC II surface expression and mRNA expression were measured. NO was induced in BMDC from both lines in response to LPS and LPS + heat stimulation; Fayoumi produced more NO with LPS treatment. Fayoumi had higher phagocytic ability and MHC II surface expression. Gene expression for the heat-related genes BAG3, HSP25, HSPA2, and HSPH1 was strongly induced with heat and few differences existed between lines. Expression for the immune-related genes CCL4, CCL5, CD40, GM-CSF, IFN-γ, IL-10, IL-12β, IL-1β, IL-6, IL-8, and iNOS was highly induced in response to LPS and different between lines. This research contributes to the sparse knowledge of genetic differences in chicken BMDC biology and function.     

Identification of the mRNA encoding interleukin-6 and its receptor,interleukin-6 receptor α, in five marsupial species

Expressed coding sequences for interleukin-6 (IL-6) and interleukin-6 receptor α (IL-6R) were examined in five marsupial species. Full length expressed coding sequences for IL-6 and IL-6R were identified and characterized in the gray short-tailed opossum (Monodelphis domestica). For IL-6, ∼225 bp fragments of the mRNA sequence were identified in the red-tailed phascogale (Phascogale calura), kultarr (Antechinomys laniger), and stripe-faced dunnart (Sminthopsis macroura), while ∼563 bp fragments of mRNA encoding IL-6R were identified in the red-tailed phascogale, kultarr, stripe-face dunnart and fat-tailed dunnart (Sminthopsis crassicaudata). Relative expression levels of IL-6 and IL-6R were examined in the heart, muscle, lung, liver, spleen and kidney of adult red-tailed phascogales, and IL-6 gene expression was found to be significantly higher in the lung and spleen than the other tissues examined, while the expression of IL-6R was significantly higher in the liver, lung and spleen. These results now serve as a reference point for examining the role and levels of IL-6 and IL-6R in the health and disease of these marsupial species. The pro-tumorigenic nature of IL-6 is of particular interest, and the identification of these IL-6 and IL-6R coding sequences provides a platform for further work to evaluate the potential role of IL-6 in marsupial cancers.