Role In Innate Immune System Research Articles

Occupational and Environmental Health Effects of Nanomaterials.

With the recent advancements in nanosciences and nanotechnologies, a large number of novel nanomaterials have been introduced in our everyday life. However, as the potential hazards of these novel nanomaterials have not yet been fully understood, concerns on their occupational and environmental health effects are mounting. In fact, recent scientific studies suggested that at least some nanoparticles actively interact with biological tissues and cause toxicological effects on the experimental animals exposed to these materials. Current research also indicates that the toxicity of manufactured nanomaterials will depend on their physical and chemical properties including their chemical composition, core size, morphology, agglomeration state, surface area, and surface charge. However, so far, quantitative understanding on the relationships between their physicochemical properties, biological toxicities, and human and environmental health effects is lacking. It is therefore imperative to have a clearer understanding of such a relationship and hence the aim of this special issue is to discuss occupational and environmental health effects of nanomaterials in relation to their various physicochemical properties. Several authors participated in this special issue to present their understanding on occupational and environmental health effects of nanomaterials. “Three-Day Continuous Exposure Monitoring of CNT Manufacturing Workplaces” by J. H. Lee et al. and “Workplace Exposure to Titanium Dioxide Nanopowder Released from a Bag Filter System” by J. H. Ji et al. are related to occupational safety and health of nanomaterial and deal with exposure assessment in the workplaces producing or handling manufactured nanomaterials. The authors of these two papers actively monitored exposure situations in the workplaces where nanomaterial manufacturing and handling were conducted and presented workers' exposure situation and exposure mitigation strategies. “Aquatic Toxicity Comparison of Silver Nanoparticles and Silver Nanowires” by E. K. Sohn et al. and “Multiwall Carbon Nanotube-Induced Apoptosis and Antioxidant Gene Expression in the Gills, Liver, and Intestine of Oryzias latipes” by J. W. Lee et al. are dealing with aquatic toxicity of one-dimensional form of nanomaterial such as wire or fiber form. E. K. Sohn et al. compared aquatic toxicity of particular forms with wire forms of nanomaterials and J. W. Lee et al. found that the gills were more sensitive to MWCNT toxicity than the other organs with gender difference and caused apoptosis with relevant gene expressions increasing caspases, while reducing expression of catalase and GST genes. Y.-H. Luo et al. studied immunotoxicity of metal-based nanoparticles showing different toxicokinetics from conventional bulk nonnanoscale materials depending on their physicochemical properties. In the paper entitled “Metal-Based Nanoparticles and the Immune System: Activation, Inflammation, and Potential Applications,” a discussion is presented on nanoparticle and innate immunity and effect of nanoparticle exposure on toll-like receptor signaling and on their role in innate immune system and effect of nanoparticle exposure on adaptive immunity. Currently, there is limited evidence on human hazards due to nanomaterial exposure. This is partly due to the very few numbers of workers or consumers exposed to nanomaterial as well as limited epidemiological or biomonitoring tools to evaluate human health hazards caused by nanomaterial exposure. L. Cabalova et al. studied micro- and nanosized particles using scanning electron microscopy and Raman microscopy in nasal mucosa from patients with chronic hypertrophic rhinosinusitis and obtained the mucosa of the inferior nasal turbinates, which are the deposition site of inhaled particles and the first barrier filtering the inhaled air. Their pilot study suggested possibility of quantification of distribution of micro- and nanosized particles in tissue samples. Their results give some promising methods by which to investigate nanoparticle exposure in human population. Nanomaterial measurement and characterization method using superresolution stimulated emission depletion (STED) microscopy presented a new method in the evaluation of nanoparticle interaction mechanisms on a cellular level. They demonstrated quantitative estimates of the number of nanoparticles administered, delivered, and internalized by epithelial cells which can be extracted from 3D STED image stacks of entire cells via image processing. This method would provide understanding on essential knowledge for risk assessment and safe design approaches in nanotechnology.

Increased expression of Toll-like receptors (TLRs) 7 and 9 and other cytokines in systemic lupus erythematosus (SLE) patients: Ethnic differences and potential new targets for therapeutic drugs

Increased expression of pro-inflammatory cytokines such as interferon, tumor necrosis factors (TNFs) and specific interleukins (ILs) has been found in a number of autoimmune diseases, including systemic lupus erythematous (SLE). These cytokines are induced by toll-like receptors (TLRs). Toll-like receptors are activated in response to accumulation of apoptotic bodies. These receptors play critical roles in innate immune systems. Increased levels of interferon-alpha (INF-α) have also been found in many SLE patients and often correlate with disease severity. The objectives of this study were to examine the expression of selected TLRs and cytokines that have been identified in animal models and some limited human studies in a group of African Americans (AA) and European Americans (EA) women with lupus in comparison to age-matched non-lupus women. Blood samples were consecutively obtained by informed consent from 286 patients, 153 lupus and 136 non-lupus, seen in the rheumatology clinics at East Carolina University. Cytokines were analyzed from blood serum using enzyme linked immunoassay (ELISA) for IL-6 and INF-α. Total RNA was isolated, using a Paxgene kit, from peripheral blood mononuclear cells of African American and European American women blood samples. Quantitative real-time PCR using the CFX real-time system was conducted on all samples to determine TLRs 7 and 9, as well as INF-α expression. Toll-like receptor 7 (p<0.01) and 9 (p=0.001) expression levels were significantly increased in lupus patients compared to age-matched controls. African American women with lupus had a 2-fold increase in TLR-9 expression level when compared to their healthy controls or European American lupus patients. However, there was no ethnic difference in expression of TLR-7 in lupus patients. INF-α expression was significantly higher in lupus patients (p<0.0001) and also showed ethnic difference in expression. Serum levels revealed significant increases in expression of IL-6, IFN-γ and TNF-α in lupus patients compared to non-lupus patients. African American women with lupus had significantly higher serum levels of IL-6 and TNF-α. African American women with lupus demonstrated increased levels of specific pro-inflammatory cytokines and Toll-like receptors when compared to EA women. Increased expression in these lupus patients provides an opportunity for targeting with antagonist as a new therapy for systemic lupus erythematous.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Lysozyme aptamer biosensor based on electron transfer from SWCNTs to SPQC-IDE

Lysozyme (Lys) is one of the most versatile and ubiquitous proteins with vital functional roles in innate immune systems, therefore its detection is of considerable importance. In this work, a sensitive and selective piezoelectric aptamer biosensor for Lys detection was constructed using an aptamer-wrapped-SWCNTs-modified interdigital electrode (IDE) which was connected to the oscillator circuit in series with piezoelectric quartz crystal (SPQC). The anti-Lys aptamer labeled with thiolate terminal was immobilized on gold surfaces of the SPQC-IDE by AuS bonding. SWCNTs were bonded to the surface of SPQC-IDE through π–π stacking between the nucleotide bases of anti-Lys aptamer and the SWCNTs sidewall. Modified SWCNTs can be substituted by Lys resulting in sensitive frequency change of SPQC-IDE. The detection limit is lower than 0.5nM for Lys, while when the biosensor was used to detect Lys concentrations in prepared human saliva sample, the recoveries were in the range of 93.1–105.4%. The novel biosensor for Lys detection proposed here is characterized by its high selectivity and sensitivity and shows great potential for use in clinical applications.

Molecular cloning and expression analysis of two β-defensin genes in the blunt snout bream (Megalobrama amblycephala)

β-Defensins are a group of cysteine-rich, cationic antimicrobial peptides that play important roles in innate immune system against pathogenic microbes invading. In this study, the part-length cDNA sequences of two β-defensin genes (maΒD-1, maΒD-2) in blunt snout bream (Megalobrama amblycephala) were identified. Homology analysis showed that the cDNA sequences of maΒD-1 and maΒD-2 had high similarities to those in common carp and zebrafish. Real-time quantitative PCR results exhibited that expression level of maΒD-1 in juvenile tissues was the highest in skin, followed by blood and liver, whereas maΒD-2 was lowly expressed in liver, kidney, brain and foregut. In the early development period, fertilized eggs to 31-day post-hatching (dph) larvae, the expression levels of maΒD-1 were higher at the stage from heart beat stage to 3 dph with the highest value at 1 dph, whereas maΒD-2 was expressed higher at fertilized eggs and late cleavage stages. Following bacterial stimulation in vivo by Aeromonas sobria, maΒD-2 expressions were significantly up-regulated in liver, skin, gill, and foregut of juveniles, and maΒD-1 expressions were significantly up-regulated in liver and skin. The results suggest that maΒD-1 and maΒD-2 may play important roles in protecting blunt snout bream embryos, fry and juveniles from pathogenic microbe invading.

Human β-Defensin 2 Is a Novel Opener of Ca 2+ -Activated Potassium Channels and Induces Vasodilation and Hypotension in Monkeys

Human β-defensin 2 (HBD2) is a cysteine-rich cationic antimicrobial peptide known for its important role in innate immune system. Intensive studies have demonstrated its antimicrobial and chemotactic activities in vitro. In this study, ELISA analysis showed that HBD2 was significantly downregulated in sera of patients with hypertension. It relaxed vessel smooth muscle by acting on the major regulatory pathways, contributing to vessel smooth muscle contraction. Electrophysiology analysis indicated that HBD2 acted as an opener of large-conductance Ca(2+)-activated potassium (BKCa)-mSlo+hβ1 channels and increased BKCa currents. Mutation analysis revealed that HBD2 activated BKCa-mSlo+hβ1 channels via interacting with Leu41 and Gln43 of β1-loop. In vivo experiments suggested that HBD2 at 4 × to 6 × of physiological concentration exerted hypotensive effect in monkeys significantly, whereas the selective blocker of BKCa channels, Paxilline, inhibited the effect. HBD2 is the first peptide opener of BKCa-mSlo+hβ1 channels. It may be a novel regulator of blood pressure and provides a new therapeutic target for the treatment of hypertension. The HBD2 blockade of the BKCa channels may represent a new type of cross-talk between immune and cardiovascular systems.

Evolutionary analysis of TLR9 genes reveals the positive selection of extant teleosts in Perciformes

The innate immune system can recognize non-self through pattern recognition receptors. Toll-like receptors were the best-known members of these receptors, and they could sense, recognize, and bind pathogen-associated molecular patterns. TLRs played an important role in innate immune system and were conserved in both invertebrate and vertebrate lineages. Thereinto, TLR9 could detect unmethylated CpG motifs in dsDNA and was expected to undergo coevolution with its microbial ligands. It was known that aquatic and terrestrial organisms dwelled in different environments which contained different pathogens, and they had to adapt to their local environmental conditions. Therefore, we collected TLR9 genes from invertebrate to vertebrate to further explore whether the huge differences between aquatic and terrestrial environments affected the TLR9s evolution between aquatic and terrestrial organisms. Molecular evolution analysis detected positively selected sites in the ancestral lineages of vertebrates, teleosts, and Perciformes but not in the ancestral lineage of mammals. In PAML, site model revealed that extant mammalian TLR9 genes underwent positive selection. However, the positive selection of extant teleosts appeared primarily in Perciformes in which there were 14 positively selected sites. Among these sites, two of them were located on the amino acid insertions of the leucine-rich repeats which could create DNA binding sites, three were found on the convex surface which might possibly affect the flexibility of the TLR solenoids, and six were located on the β-face of concave surface which contained the ligand-binding sites of the TLR solenoids. In other ML methods, we also found three sites under selection that coincided with the codons identified by M8 and these sites were all located in LRRs. The diverse aquatic and terrestrial environments might possess different pathogens to make the living organisms adapt to their local environmental conditions. The positive selection on LRRs in TLR9s of Perciformes might be associated with the adaptation to the rapidly evolving pathogens in the water.

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity.

Role of Innate Immune System in Inflammation and Cardiac Remodeling After Myocardial Infarction

The innate immune system is well recognized as the first line defense of foreign pathogens; however, it can also recognize endogenous signals released from injured tissues and induce sterile inflammation. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) have been identified as its receptors, and they have been shown to play a key role in the disease processes of sterile inflammation, including myocardial infarction (MI). In particular, NLRs are the key components of the caspase-1 activating platform known as the "inflammasome" which produces the potent proinflammatory cytokine interleukin-1β. The current article reviews the role of the innate immune system, especially TLRs and inflammasomes, in the pathophysiology of MI.

MRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

BackgroundThe Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis.ResultsA database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed.ConclusionsThis study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Expression pattern, promoter activity and bactericidal property of β-defensin from the mandarin fish Siniperca chuatsi

β-Defensin (BD) are cysteine-rich, cationic antimicrobial peptides which play an important role in innate immune system against invading microbes. In the present study, the cDNA cloning, expression analysis, transcriptional regulation and antimicrobial activity of β-defensin (ScBD) from mandarin fish (Siniperca chuatsi) were characterized. The cDNA sequence of ScBD is 596 bp which encodes a protein of 63 amino acids (aa). The ScBD gene comprises three exons and two introns. The signal peptide is located in the first exon. ScBD contains 6 cysteines, and belongs to fish defensin 2 group based on phylogenetic analysis. Real-time quantitative PCR results showed that the mRNA transcripts of ScBD were distributed mainly in mucosal and lymphoid organs/tissues including intestine, gill, head kidney, kidney and spleen, with the highest level observed in spleen. Western blotting analysis revealed that the ScBD protein was abundant in head kidney, gill and spleen. A total of 3268 bp 5′ flanking region of the ScBD gene promoter was sequenced, which contained a number of putative transcriptional binding sites for transcription factors. These transcription factors were analyzed using in vitro luciferase assay. The DNA region from position of −705 to −498 bp contains positive regulatory elements and that of −227 to +54 bp harbors the TATA which is essential for initiating gene expression. In addition, the ScBD peptide showed antibacterial activity against Escherichia coli M15, Staphylococcus aureus and Aeromonas hydrophila, whilst no effect on Edwardsiella tarda. These data suggest that the ScBD is importantly involved in host immune responses to invasion of bacterial pathogens.

Protective Role of Interferon Regulatory Factor 3-Mediated Signaling against Prion Infection

Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP(Sc) was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP(Sc) formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.

Cloning and expression analysis of interferon regulatory factor (IRF) 3 and 7 in large yellow croaker, Larimichthys crocea

The interferon regulatory factor (IRF)3 and IRF7 are considered to play essential roles in innate immune system’s antiviral responses. In this report, the full-length cDNA and genomic structure and immune response characterizations of IRF3 and IRF7 were investigated in large yellow croaker, Larimichthys crocea. The full-length cDNA of L. crocea (Lc)IRF3 was of 2204 bp, including a 5′-terminal untranslated region (UTR) of 41 bp, a 3′-terminal UTR of 774 bp and an open reading frame (ORF) of 1389 bp encoding a polypeptide of 462 amino acids residues. The full-length cDNA of LcIRF7 was of 1979 bp, including a 5′-terminal UTR of 47 bp, a 3′-terminal UTR of 636 bp and an ORF of 1296 bp encoding a polypeptide of 431 amino acids. The putative amino acid sequence of both LcIRF3 and LcIRF7 contained a typical IRF domain at the N-terminal and an IRF3 domain at the C-terminal. Furthermore, we obtained 4517 nucleotides (nt) LcIRF3 genome sequence based on the full-length cDNA, which contained 11 exons and 10 introns. The full-length genome sequence of LcIRF7 was of 3991 nucleotides, including 9 exons and 8 introns. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of LcIRF3 and LcIRF7 with the most predominant expression of LcIRF3 and LcIRF7 in the liver and in the gill, respectively. The expression levels of LcIRF3 and LcIRF7 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were tested in blood, spleen and liver. The results showed that the highest relative expression of LcIRF3 was in the liver at 24 h after poly I:C injection with 90 times greater than that of the non-injection group (p < 0.05). Moreover, LcIRF3 transcription increased significantly at most time point in blood and spleen tissue after poly I:C stimulation compared with that of the control group. After LPS injection, the peak value of LcIRF7 was in the liver with 207 times (at 3 h) as much as that in the control group (p < 0.05). In addition, LcIRF7 expression was significantly induced by poly I:C injection in spleen. Both LcIRF3 and LcIRF7 transcripts did not show significant change after V. parahaemolyticus stimulation. These results indicated that IRF3 and IRF7 might play an important role in large yellow croaker’s defense against viral and bacterial infection.

Noninvasive molecular imaging of interferon beta activation in mouse liver

Interferon beta (IFN-β) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-β activation in mouse liver by noninvasive molecular imaging. Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-β. Luciferase activity was used as an indicator of the IFN-β promoter activity in vitro and in vivo. The expression level of IFN-β in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-β in the sera was not induced by MHV-3. Moreover, IFN-β activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-β production in the sera. Therefore, a novel visual method to monitor IFN-β promoter activity was established in the current study. This novel sensitive method can be used for not only assessing IFN-β activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-β production.

Immunologic markers associated with favorable prognosis in breast cancer patients: Role of innate immune system.

43 Background: Recognition of tumor cells by NK cells is mediated by the interaction of activating and inhibitory NK cell receptors with ligands expressed on the interacting cell. In addition to inhibitory and activating receptors, NK cells express adhesion molecules that allow conjugate formation between NK cells and their tumor targets. We have recently identified by transcriptional analysis of breast tumor specimens a signature of 5 genes, which included B cell response and interferon signaling, found to predict with &gt; 85% accuracy relapse-free survival. In the current study we analyzed by gene expression profile, whether a differential expression of activatory and inhibitory receptors and gene involved in NK cell-target interactions could be identified in relapse and relapse free patients.Methods: RNA extraction and gene expression analysis was performed on tumor specimens with at least 10% of infiltrating cells deriving from 9 from breast cancer patients who had either 3-7 years relapse-free survival (n=9) or developed tumor relapse within 1-3 years after the initial treatment (n=8). Data were analyzed by BRB tools and Partek software.Results: Our results showed that patients with a favorable prognosis showed increased expression of genes involved in NK interaction with tumor cells and NK activating signaling. In particular an up-regulation of leukocyte function-associated antigen 1 (LFA-1) gene, typically involved in NK cells adhesion to target cells and DNAM 1, usually associated with activation of NK cells and considered one of the major protagonist of NK- Dendritic cells crosstalk, was observed to occur in relapse free patients. In addition, an up regulation of CD96 and CRTAM which, like DNAM 1, promote NK cell-target cell adhesion by interacting with the Necl2 and poliovirus receptor (PVR), was observed in relapse free patients. Conclusions: Our observation suggests that the NK signatures are associated with favorable outcome in breast cancer and allow us to generate new hypothesis on the role of innate immunity in this contest.

The influence of oocyte cortisol on the early ontogeny of intelectin and TLR-5, and changes in lysozyme activity in rainbow trout (Oncorhynchus mykiss) embryos

The ontogeny of lysozyme activity, intelectin, TLR-5M and TLR-5S gene expression and intelectin localization was examined in rainbow trout (Oncorhynchus mykiss) reared from oocytes immersed for 3h prior to fertilization in either ovarian fluid alone (CC) or cortisol-enriched ovarian fluid at either 100 ng mL(-1) (C1) or 1000 ng mL(-1) (C2) [final oocyte cortisol concentrations were ~3, ~5, and ~7.5 ng oocyte(-1) for the CC, C1 and C2 treatment groups, respectively]. Lysozyme activity was elevated in the cortisol-treated groups from the zygote until 13-days post fertilization (dpf), but was not affected at 21-dpf. Intelectin levels were elevated in both cortisol treatment groups at 12-hpf (2-cell stage) and then suppressed between 36- and 48-hpf. Intelectin mRNA transcript levels were elevated in both cortisol treatment groups in oocytes; there were no differences among treatment groups at 1- and 5-dpf, and suppressed in the C2 treatment group in 13-dpf and 26-dpf. TLR-5 mRNA transcripts were higher in cortisol-treated oocytes prior to fertilization; TLR-5S mRNA was more abundant than TLR-5M mRNA. The ontogeny of the gene expression patterns, and the gene, lectin and lysozyme responses to oocyte cortisol adjustments suggest an important role of innate immune systems in the early cleavage stages of embryonic cells.

A double WAP domain-containing protein PmDWD from the black tiger shrimp Penaeus monodon is involved in the controlling of proteinase activities in lymphoid organ

A homolog of mammalian secretory leucocyte proteinase inhibitor or SLPI known as a double WAP domain (DWD) protein has been found in penaeid shrimp and believed to play an important role in innate immune system of the shrimp. The PmDWD identified from the Penaeus monodon EST database was investigated for its expression under pathogen infection. Infections by Vibrio harveyi and white spot syndrome virus (WSSV) up-regulated the expression of the PmDWD, which was peaked at about 24 h post infection and, then, subsided to more or less normal level. The PmDWD was expressed in various tissues of normal, 24-h WSSV-injected and leg-amputated shrimp, predominantly in the hemocytes. The expression was dramatically increased in lymphoid organ upon WSSV infection and leg amputation. The recombinant PmDWD (r PmDWD) was not active against the commercial proteinases: trypsin, chymotrypsin, elastase and subtilisin while its mutant r PmDWD_F70R was active against the subtilisin. By using agar diffusion assay, the r PmDWD inhibited the crude proteinases from lymphoid organs of leg-amputated and WSSV-infected shrimp. It inhibited the crude proteinases from Bacillus subtilis as well. Unlike the mammalian SLPIs, the r PmDWD had no antimicrobial activity against various bacteria.

The marine medaka Oryzias melastigma – A potential marine fish model for innate immune study

The objective of this study is to develop the marine medaka Oryzias melastigma as a potential marine fish model for innate immune and immunotoxicological studies. Hepcidin plays an important role in innate immune system. Two hepcidin genes (OM-hep1 and OM-hep2) were identified and characterized in the O. melastigma, which were highly conserved with other reported hepcidins. During embryogenesis, significant elevation of OM-hep1 and OM-hep2 transcripts were coincided with liver development in the embryos. In adult medaka, differential tissue expressions of both hepcidin transcripts were evident: high in liver, moderate in spleen and low in non-immune tissues. After bacterial challenge, the two hepcidin mRNAs were rapidly and remarkably induced in liver and spleen, suggesting the two OM-hepcidins in O. melastigma play a complementary role in innate defense. Gender difference in time of induction and extent of the two hepcidin mRNAs elevation in infected O. melastigma should be considered in immunotoxicological studies.

Aflatoxin B1 and its toxic effects on immune response of teleost fishes: a review

Aflatoxins represent, among all known mycotoxins, the best characterised and most investigated foodborne contaminants at present. This importance led to early research on acute and chronic toxicity. Further research showed that chronic toxicity due to aflatoxins could cause silent damages, causing the more evident carcinomas, being immunotoxicity one of the first toxicological effects studied on both mammals and aquatic farmed species, such as fishes. Although many of the aflatoxin immunotoxicity scientific information has been generated a quarter of century ago, the recent role of innate immune system on aquatic species still demands for up-to-date results, and expresses that the research on immunotoxicity caused by aflatoxins is yet to be completed. Based on these considerations, the aims of this review are to collect existing data on toxic effects on immune response of teleost fishes due to aflatoxins that could give us a perspective for the follow up research, and to gain new insights on the importance caused by the consumption of aflatoxin contaminated fish through the trophic chain.

Anti-inflammatory actions of Syk inhibitors in macrophages involve non-specific inhibition of toll-like receptors-mediated JNK signaling pathway

Toll-like receptors (TLRs) are a major family of pattern recognition receptors (PRRs) and play a crucial role in innate immune system. Even though non-receptor spleen tyrosine kinase (Syk) is a key signaling molecule of immunoreceptor tyrosine-based activation motifs-containing immunoreceptors, its role in TLRs signaling is not clearly understood. Herein, we investigated the role of Syk in TLR-mediated signaling and gene regulation. In bone marrow-derived macrophages (BMDMs) and RAW 264.7 macrophages, treatment of poly(I:C), LPS and CpG, which are specific ligands of TLR3, TLR4 and TLR9, respectively, can increase the mRNA levels of several pro-inflammatory cytokines and mediators, including IFNβ, TNFα, MIP2, IL-6, IL-12β, iNOS and COX-2. The gene upregulation caused by TLR was inhibited by Syk inhibitor (SykI) and JNK inhibitor (SP600125). Accordingly we found the abilities of TLR3, TLR4 and TLR9 ligands to induce Syk and JNK activation, as evidenced by increased Syk autophosphorylation on Y519/Y520, JNK phosphorylation and both kinase activities. We also found that TLRs-mediated JNK activation, but not IKK, p38 and ERK activation as well as IκB degradation in BMDM and RAW 264.7 cells, was blocked by SykI. Nevertheless TLR-mediated JNK activation as well as the increased protein expression of iNOS and COX-2 remained unchanged when Syk protein was knockdown by siRNA approach. With in vitro kinase assay we found two commercial Syk inhibitors (SykI, and BAY61-3606) have direct inhibition on JNK activity. These findings demonstrate that the non-selective action of SykI on JNK should be taken into consideration upon using them to explore the biological actions of Syk.