Silkworm ferritin 1 heavy chain homolog is involved in defense against bacterial infection through regulation of haemolymph iron homeostasis

Iron functions as a nutrient and a potential toxin in all organisms. It plays a key role in the interaction between microbes and their hosts as well. Microbial infection disrupts iron homeostasis in the host; meanwhile the host endeavors to keep the homeostasis through iron transport and storage. Transferrins and ferritins are the major iron-binding proteins that affect iron distribution in insects. In this study, we investigated a possible involvement of Bombyx mori ferritin 1 (BmFer1) heavy chain homolog in the defense against bacterial infection in the silkworm larvae. The BmFer1 mRNA abundance was up-regulated in hemocytes, but not in fat body, after Pseudomonas aeruginosa or Staphylococcus aureus infection. The infection resulted in elevated iron levels in the hemolymph. Injection of recombinant BmFer1 protein into hemocoel reduced the plasma iron level after infection, limited the bacterial growth in the hemolymph, and resulted in a lower mortality caused by infection. Our study indicated that B. mori ferritin-1 may restrict iron access of the invading bacteria to block their growth as a defense strategy.     

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.     

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.     

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.     

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.     

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.     

Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.     

Expansion and differentiation of IgM+ B cells in the rainbow trout peritoneal cavity in response to different antigens

To date, intraperitoneal (i.p.) injection seems to be the most effective vaccination route in aquaculture, as many i.p. administered fish vaccines are capable of conferring strong and long-lasting immune responses. Despite this, how peritoneal leukocytes are regulated upon antigen encounter has only been scarcely studied in fish. Although, in the past, myeloid cells were thought to be the main responders to peritoneal inflammation, a recent study revealed that IgM⁺ B cells are one of the main cell types in the teleost peritoneal cavity in response to pathogenic bacteria. Thus, in the current work, we have focused on establishing how IgM⁺ B cells are recruited into the peritoneum in rainbow trout (Oncorhynchus mykiss) comparing different antigens: Escherichia coli as a bacterial model, E. coli-derived lipopolysaccharide (LPS) or viral hemorrhagic septicemia virus (VHSV). In addition to studying their capacity to dominate the peritoneal cavity, we have established how these IgM⁺ B cells are regulated in response to the different antigens, determining their levels of IgM secretion, surface MHC II expression, cell size and phagocytic abilities. Our results reveal that IgM⁺ B cells are one of the main cell types amplified in the peritoneum in response to either bacterial or viral antigens and that these immunogenic stimulations provoke a differentiation of some of these cells towards plasmablasts/plasma cells whereas others seem to be implicated in antigen presentation. These findings contribute to a better understanding of the immune processes that regulate peritoneal inflammation in teleost fish.     

High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans

Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4⁺ Effector Memory T cells. In human CD4⁺ T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4⁺ T cells, eIF6 levels control interferon-γ (IFN−γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery.     

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study.     

The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.     

Recombinant fragilysin isoforms cause E-cadherin cleavage of intact cells and do not cleave isolated E-cadherin

The fragilysin (BFT) is a protein secreted by enterotoxigenic Bacteroides fragilis strains. BFT contains zinc-binding motif which was found in the metzincins family of metalloproteinases. In this study, we generated three known recombinant isoforms of BFT using Escherichia coli, tested their activity and examined whether E-cadherin is a substrate for BFTs. BFT treatment of HT-29 cells induced endogenous E-cadherin cleavage, and this BFT activity requires the native structure of zinc-binding motif. At the same time recombinant BFTs did not cleave recombinant E-cadherin or E-cadherin in isolated cell fractions. It indicates that E-cadherin may be not direct substrate for BFT. We also detected and identified proteins released into the cultural medium after HT-29 cells treatment with BFT. The role of these proteins in pathogenesis and cell response to BFT remains to be determined.     

Characterization and antiviral activity of a newly identified defensin-like peptide,HEdefensin, in the hard tick Haemaphysalis longicornis

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.     

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared “poised” and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.     

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.     

Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS^E), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT^®. A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.     

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 10⁹ heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.     

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.