Stimulator of interferon genes from Asian swamp eel (MaSTING) is involved in host defense against bacterial infection.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated protein that plays critical roles in innate immunity and pathogenesis of various diseases. To date, teleost STING against viral stimulation has been identified, whereas STING signaling events in fish against bacteria are not well understood. In the present study, the open reading frame (ORF) of STING from Asian swamp eel (Monopterus albus) was cloned (named MaSTING) and its roles in bacterial infection were investigated. Amino acid sequence alignment and phylogenetic analysis revealed that MaSTING had conserved structures with mammalian STING and shared the closest relationship with mandarin fish STING. Subcellular localization analysis showed that MaSTING distributed in the whole cytoplasm and mainly co-localized with ER. Expression pattern analysis found that MaSTING was constitutively expressed in all the examined tissues with the highest expression in the liver and spleen. Post stimulation with bacteria and various PAMPs, the expression of MaSTING was induced at indicated time points in the immune-related organs and isolated peripheral blood leucocytes. Furthermore, the mechanism underlying MaSTING against bacterial infection was further studied. The qPCR analysis showed that MaSTING overexpression promoted 2'3'-cGAMP induced the expression of IFN-1, ISG15, Viperin, Mx, IL-1ß and TNF-α. Western blotting assay suggested that MaSTING significantly enhanced the phosphorylation of TANK-binding kinase 1 (TBK1) and p65. MaSTING also significantly increased the luciferase activity of IFN-1 and NF-κB promoters. Taken together, MaSTING is involved in host defense against bacterial infection by inducing the inflammatory response.

Identification and functional analysis of Mannose receptor in Asian swamp eel (Monopterus albus) in response to bacterial infection.

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely MaMR). The open reading frame of MaMR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that MaMR shared the highest similarity with that of Paralichthys olivaceus. The expression of MaMR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda, the transcript level of MaMR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of MaMR was significantly induced post stimulated with LPS and LTA. Then the MaMR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that rMaMR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that MaMR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1ß and tnf-α in EPC cells. Taken together, these results reveal that MaMR plays an important role in the immune response of fish to pathogen infections.

Activation of the NLRP1 inflammasome and its ligand recognition in the antibacterial immune response of common carp (Cyprinus carpio).

NLRP1 (NLR family pyrin domain containing 1) is the first member of NOD-like receptors (NLRs) which can form inflammasome and play critical roles in innate immunity and pathogenesis of various diseases. To date, many NLRs and inflammasome-related genes have been identified in teleost, however, the activation of NLRP1 inflammasome is only found in zebrafish, and the activator of fish NLRP1 is unclear. In the present study, the activation of CcNLRP1 inflammasome and its function in innate immune defence of common carp was investigated. The expression of CcNLRP1 was induced in immune-related tissues of common carp upon challenge with Edwardsiella tarda and Aeromonas hydrophila. The colocalization of CcNLRP1 and CcASC, ASC oligomerization, and interaction between CcNLRP1CARD and CcASC was observed in 293T, Hela and EPC cells, suggesting that the CcNLRP1 inflammasome was activated in common carp. Furthermore, we found that MDP may be the specific ligand of CcNLRP1, which can activate the CcNLRP1 inflammasome. Taken together, the present study identifies a new inflammasome in common carp, and is beneficial to the control of infectious diseases in carp farming.

Molecular characterization of a new IgZ3 subclass in common carp (Cyprinus carpio) and comparative expression analysis of IgH transcripts during larvae development.

BACKGROUND: Immunoglobulins (Igs) distributed among systemic immune tissues and mucosal immune tissues play important roles in protecting teleosts from infections in the pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have different immune functions. RESULTS: In the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio). This gene exhibited a different tissue-specific expression profile than the reported genes IgZ1 and IgZ2. The obtained IgZ-like subclass gene designated CcIgZ3, had a complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. Phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as IgZ/IgT genes of other teleosts. Basal expression detection of the immunoglobulin heavy chain (IgH) in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and mucosal-associated lymphoid tissues. CcIgZ3 was expressed at the highest levels in the head kidneys, gills, and gonads, followed by the spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood; it was expressed at a very low level in the skin. The transcript expression of CcIgZ3 in leukocytes isolated from peripheral blood cells was significantly higher than that in leukocytes isolated from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection or immersion. RT-qPCR analysis demonstrated that significant differences in CcIgZ3 mRNA levels existed between the immersion and injection groups in all the examined tissues, including the head kidney, spleen, liver, and hindgut; in particular, the CcIgZ3 mRNA level in the hindgut was higher in the immersion group than in the injection group. The different routes of A. hydrophila exposure in common carp had milder effects on the IgM response than on the CcIgZ3 response. Further study of the relative expression of the IgH gene during the development of common carp showed that the tissue-specific expression profile of CcIgZ3 was very different from those of other genes. RT-qPCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually in common carp during the early larval development stage from 1 day post fertilization (dpf) to 31 dpf with a dynamic tendency similar to those of IgZ1 and IgZ2, and IgM was the dominant Ig with obviously elevated abundance. Analyses of the tissue-specific expression of IgHs in common carp at 65 dpf showed that CcIgZ3 was expressed at mucosal sites, including both the hindgut and gill; in contrast, IgZ1 was preferentially expressed in the hindgut, and IgZ2 was preferentially expressed in the gill. In addition to RT-qPCR analysis, in situ hybridization was performed to detect CcIgZ3-expressing cells and IgM-expressing cells. The results showed that CcIgZ3 and IgM transcripts were detectable in the spleens, gills, and hindguts of common carp at 65 dpf. CONCLUSIONS: These results reveal that CcIgZ3 gene transcripts are expressed in common carp during developmental stage not only in systemic tissues but also in mucosal tissues. CcIgZ3 expression can be induced in immune tissues by A. hydrophila challenge via immersion and intraperitoneal injection with significantly different expression profiles, which indicates that CcIgZ3 is involved in the antimicrobial immune response and might play an important role in gut mucosal immunity.

Molecular characterization and immune functional analysis of IRF2 in common carp (Cyprinus carpio L.): different regulatory role in the IFN and NF-κB signalling pathway.

BACKGROUND: Interferon regulatory factor 2 (IRF2) is an important transcription factor, which can regulate the IFN response and plays a role in antiviral innate immunity in teleost. RESULTS: In the present study, the full-length cDNA sequence of IRF2 (CcIRF2) was characterized in common carp (Cyprinus carpio L.), which encoded a protein containing a conserved DNA-binding domain (DBD) and an IRF-associated domain (IAD). Phylogenetic analysis showed that CcIRF2 was most closely related with IRF2 of Ctenopharyngodon idella. CcIRF2 transcripts were detectable in all examined tissues, with higher expression in the gills, spleen and brain. CcIRF2 expression was upregulated in immune-related tissues of common carp upon polyinosinic:polycytidylic acid (poly (I:C)) and Aeromonas hydrophila stimulation and induced by poly (I:C), lipopolysaccharide (LPS), peptidoglycan (PGN) and flagellin in the peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs). In addition, overexpression of CcIRF2 decreased the expression of IFN and IFN-stimulated genes (ISGs), and a dual-luciferase reporter assay revealed that CcIRF2 could increase the activation of NF-κB. CONCLUSIONS: These results indicate that CcIRF2 participates in antiviral and antibacterial immune response and negatively regulates the IFN response, which provide a new insight into the regulation of IFN system in common carp, and are helpful for the prevention and control of infectious diseases in carp farming.

Cutting Edge: TRAF6 Mediates TLR/IL-1R Signaling-Induced Nontranscriptional Priming of the NLRP3 Inflammasome.

NLRP3 inflammasome activiation requires two sequential signals. The priming signal 1 from TLRs or cytokine receptors induces the transcription of NLRP3 and IL-1ß, and concomitantly promotes transcription-independent activation of caspase-1. The activating signal 2 can be provided by microbial products or danger signals. In this study we found that TRAF6 is necessary for the nontranscriptional priming of NLRP3 inflammasome by TLR/IL-1R derived signals. Deficiency of TRAF6 specifically inhibited TLR/IL-1R priming-initiated caspase-1 cleavage, pyroptosis, and secretion of presynthesized IL-18. Mechanistically, TRAF6 promoted NLRP3 oligomerization as well as the interaction between NLRP3 and apoptosis-associated speck-like protein containing a CARD. Of note, the nontranscriptional priming via TRAF6 did not involve mitochondrial reactive oxygen species or the phosphorylation of Jnk, Erk, and Syk, whereas the ubiquitin E3 ligase activity of TRAF6 was required. Our findings thus extended cognition on the mechanism of NLRP3 inflammasome activation, and provided a novel target for controlling NLRP3-related diseases.

Hyperactivation of the NLRP3 Inflammasome in Myeloid Cells Leads to Severe Organ Damage in Experimental Lupus.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome associated with severe organ damage resulting from the activation of immune cells. Recently, a role for caspase-1 in murine lupus was described, indicating an involvement of inflammasomes in the development of SLE. Among multiple inflammasomes identified, the NLRP3 inflammasome was connected to diverse diseases, including autoimmune encephalomyelitis. However, the function of NLRP3 in SLE development remains elusive. In this study, we explored the role of NLRP3 in the development of SLE using the pristane-induced experimental lupus model. It was discovered that more severe lupus-like syndrome developed in Nlrp3-R258W mice carrying the gain-of-function mutation. Nlrp3-R258W mutant mice exhibited significantly higher mortality upon pristane challenge. Moreover, prominent hypercellularity and interstitial nephritis were evident in the glomeruli of Nlrp3-R258W mice. In addition, hyperactivation of the NLRP3 inflammasome in this mouse line resulted in proteinuria and mesangial destruction. Importantly, all of these phenotypes were largely attributed to the Nlrp3-R258W mutation expressed in myeloid cells, because Cre recombinase-mediated depletion of this mutant from such cells rescued mice from experimental lupus. Taken together, our study demonstrates a critical role for NLRP3 in the development of SLE and suggests that modulating the inflammasome signal may help to control the inflammatory damage in autoimmune diseases, including lupus.

Molecular characterization and functional analysis of interferon regulatory factor 9 (irf9) in common carp Cyprinus carpio: a pivotal molecule in the Ifn response against pathogens.

In the present study, interferon (IFN) regulatory factor (IRF) 9 gene (irf9) was identified and characterized in common carp Cyprinus carpio. The predicted protein sequence of Irf9 contains a DNA binding domain (DBD) that possess five tryptophans, an IRF association domain (IAD) and two nuclear localisation signals (NLS). Alignment of Irf9 of C. carpio with the corresponding Irf9 proteins of other species showed that the DBD is more highly conserved than the IAD. The putative Irf9 protein sequence of C. carpio shares higher identities with teleosts (53.8-82.3%) and lower identities with mammals (30.2-31.0%). Phylogenetic studies of the putative amino-acid sequence of IRF9 based on the neighbour-joining method showed that Irf9 of C. carpio has the closest relationship with the grass carp Ctenopharyngodon idella. Tissue distribution analysis showed that irf9 transcripts were detectable in all examined tissues with the highest expression in the skin and the lowest expression in the head kidney. Poly I:C and Aeromonas hydrophila stimulation up-regulated irf9 expression in the spleen, head kidney, foregut and hindgut at different time intervals. In addition, irf9 was induced by Poly I:C and lipopolysaccharides (LPS) in vitro. These results indicate that Irf9 participates in antiviral and antibacterial immunity. Transfection of irf9 up-regulated the expression of cytokines, including type I IFN, protein kinase R (PKR), interferon-stimulated gene (ISG)15 and tumour necrosis factor (TNF)α in epithelioma papulosum cyprini cells (EPC) upon poly I:C and LPS stimulation. A dual-luciferase reporter assay revealed that Irf9 has no effect on NF-κB activation. The present study on Irf9 provides new insights into the IFN system of C. carpio and a valuable experimental platform for future studies on the immune system of fish.

Non-mammalian Toll-like receptor 18 (Tlr18) recognizes bacterial pathogens in common carp (Cyprinus carpio L.): Indications for a role of participation in the NF-κB signaling pathway.

Toll-like receptors are important pattern recognition receptors that can recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity. In the present study, tlr18 was identified from common carp (Cyprinus carpio L.) (named Cctlr18). The deduced amino acid sequence contained only a signal peptide, eight LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/IL-1 receptor) domain. Phylogenetic analysis showed that CcTlr18 was most closely related to Ctenopharyngodon idella Tlr18. Quantitative real-time PCR analysis showed that Cctlr18 was constitutively expressed in all investigated tissues with the highest expression level in the skin and lowest expression in the gonad. After injection with inactivated Aeromonas hydrophila, Cctlr18 expression was significantly up-regulated in the head kidney, foregut, hindgut and skin. Moreover, significant up-regulation of Cctlr8 was observed in the spleen, head kidney, hindgut and skin after immersion with live A. hydrophila. In addition, the expression of Cctlr18 was up-regulated in PGN or flagellin-stimulated HKLs. Luciferase reporter assays showed that Cctlr18 activated NF-κB in 293 T cells and that NF-κB activity was enhanced in Cctlr18 and Ccmyd88 co-transfected cells. Furthermore, Cctlr18 could induce the expression of cytokines genes, including ifn, il-1ß and il-10, in EPC cells. The results suggested that Cctlr18 plays an important role in the immune response and provides basic information for investigating the mechanisms of fish tlr18.

Molecular characterization and expression pattern of X box-binding protein-1 (XBP1) in common carp (Cyprinus carpio L.): Indications for a role of XBP1 in antibacterial and antiviral immunity.

X box-binding protein-1 (XBP1) is a transcription factor that is essential for the unfolded protein response (UPR) and the differentiation of plasma cells, and some findings have also uncovered its function in innate immunity. XBP1 typically has two different transcripts, un-spliced (XBP1u) and spliced forms (XBP1s), but XBP1s is an active transcription factor in the regulation of target genes. To date, there is no evidence about the identification and function of XBP1 in common carp. Moreover, no data are currently available regarding the role of fish XBP1 in innate immunity. Thus, to determine whether XBP1 is involved in innate immune response in common carp, we cloned CcXBP1s and examined the expression of XBP1s and a XBP1s stimulated gene (IL-6) after Aeromonas hydrophila (A. hydrophila) and polyinosinic-polycytidylic acid (polyI:C) challenges. The results imply that CcXBP1s, as an active transcription factor, might play regulation roles in the antibacterial and antiviral innate immune responses of common carp. This allows us to gain new insights into the immunological function of XBP1 in fish innate immunity and the evolution of this important class of genes across vertebrates.

Molecular characterization of a fish-specific toll-like receptor 22 (TLR22) gene from common carp (Cyprinus carpio L.): Evolutionary relationship and induced expression upon immune stimulants.

In the host innate immune system, various pattern recognition receptors (PRRs) recognize conserved pathogens-associated molecular patterns (PAMPs), and represent an efficient first line of defense against invading pathogens. TLR22 is one of the fish-specific Toll-like receptors (TLRs), identified in a variety of fish species. In this study, we report the cloning and identification of a TLR22 cDNA from the gills of common carp (Cyprinus carpio L.). The full-length CcTLR22 cDNA was 3301 bp long, including a 32 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 2838 bp and a 432 bp 3'-UTR.The CcTLR22 protein was found to comprise a signal peptide, 16 LRR domains, a LRRCT domain in the extracellular region and a TIR domain in the cytoplasmic region, which fits with the characteristic TLR domain architecture. The genomic organization of CcTLR22 was identified, which was encoded by an uninterrupted exon. Sequence alignment and phylogenetic analysis showed that all known teleost TLR22 members were clustered into an independent clade of the TLR22 family, and showed high amino acid identities with other fish TLRs. Real-time PCR assay showed that CcTLR22 mRNA was expressed in almost all tissues examined, while the levels obviously varied among different tissues. When challenged with poly(I:C) (a viral model) or A. hydrophila bacteria, the expression level of CcTLR22 was up-regulated in a variety of common carp tissues. These results indicate that CcTLR22 plays a significant role in systemic as well as mucosal defence after viral or bacterial stimulation or infection.

Pathogen invasion changes the intestinal microbiota composition and induces innate immune responses in the zebrafish intestine.

Numerous bacteria are harbored in the animal digestive tract and are impacted by several factors. Intestinal microbiota homeostasis is critical for maintaining the health of an organism. However, how pathogen invasion affects the microbiota composition has not been fully clarified. The mechanisms for preventing invasion by pathogenic microorganisms are yet to be elucidated. Zebrafish is a useful model for developmental biology, and studies in this organism have gradually become focused on intestinal immunity. In this study, we analyzed the microbiota of normal cultivated and infected zebrafish intestines, the aquarium water and feed samples. We found that the predominant bacteria in the zebrafish intestine belonged to Gammaproteobacteria (67%) and that feed and environment merely influenced intestinal microbiota composition only partially. Intestinal microbiota changed after a pathogenic bacterial challenge. At the genus level, the abundance of some pathogenic intestinal bacteria increased, and these genera included Halomonas (50%), Pelagibacterium (3.6%), Aeromonas (2.6%), Nesterenkonia (1%), Chryseobacterium (3.4‰), Mesorhizobium (1.4‰), Vibrio (1‰), Mycoplasma (0.7‰) and Methylobacterium (0.6‰) in IAh group. However, the abundance of some beneficial intestinal bacteria decreased, and these genera included Nitratireductor (0.8‰), Enterococcus (0.8‰), Brevundimonas (0.7‰), Lactococcus (0.7‰) and Lactobacillus (0.4‰). Additionally, we investigated the innate immune responses after infection. ROS levels in intestine increased in the early stages after a challenge and recovered subsequently. The mRNA levels of antimicrobial peptide genes lectin, hepcidin and defensin1, were upregulated in the intestine after pathogen infection. These results suggested that the invasion of pathogen could change the intestinal microbiota composition and induce intestinal innate immune responses in zebrafish.

Expression profile of carp IFN correlate with the up-regulation of interferon regulatory factor-1 (IRF-1) in vivo and in vitro: the pivotal molecules in antiviral defense.

Interferon regulatory factors (IRFs) are a family of transcription factors that mediate the transcriptional regulation of interferon (IFN) genes and IFN-inducible genes. In this study, IRF-1 gene is cloned from the common carp, Cyprinus carpio L., named CcIRF-1. The full-length cDNA of CcIRF-1 is 1427 bp, including an open reading frame of 861 bp encoding a protein of 286 amino acids. The putative CcIRF-1 is characterized by a conserved DNA-binding domain and includes a signature of six conserved tryptophan residues. The genomic sequence of CcIRF-1 is described, which consists of 9 exons and 8 introns. The sequence analysis shows that CcIRF-1 is clustered into IRF-1 subfamily, and has the closest relationship with the zebrafish IRF-1. CcIRF-1 is found constitutively expressed in different organs of healthy common carp. The main findings are that CcIRF-1 is up-regulated following stimulation with poly(I:C) in all tested tissues. Moreover, the downstream gene of IRF-1 - IFN is found to be correlated with the up-regulation of IRF-1 after injection with poly(I:C). Furthermore, we also isolate the peripheral blood leukocytes (PBLs) and find that there is a relevance between the expression profile of CcIRF-1 and IFN in poly(I:C) stimulated PBLs.

Genome analysis of enterovirus 71 strains differing in mouse pathogenicity.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) and is occasionally associated with severe neurological diseases. The investigation of virulence determinants of EV71 is rudimentary. Therefore, it is important to understand the relationship between EV71 virulence and genomic information. In this study, a series of analyses about full-length genomic sequence were performed on six EV71 strains isolated from HFMD patients with either severe or mild clinical symptoms. A one-day-old BALB/c mouse model was used to study the infection characteristics. Results showed all six strains were of the subgenogroup C4a. Viral full-length genomic sequence analysis showed that a total of 40 nucleotide differences between strains of highly and low virulence were revealed. Among all mutations, three nucleotide mutations were found in the untranslated region. A mutation, nt115, at internal ribozyme entry site (IRES) caused RNA secondary structural change. The other 37 mutations were all located in the open reading frame resulting in 8 amino acid mutations. Importantly, we discovered that a mutation of amino acid (Asn1617 → Asp1617) in the 3C proteinase (3C(pro)) of highly and low pathogenic strains could lead to conformational change at the active center, suggesting that this site may be a virulence determinant of EV71.

Characterization of common carp (Cyprinus carpio L.) interferon regulatory factor 5 (IRF5) and its expression in response to viral and bacterial challenges.

BACKGROUND: Common carp (Cyprinus carpio L.), one of the most economically valuable commercial farming fish species in China, is often infected by a variety of viruses. As the first line of defence against microbial pathogens, the innate immune system plays a crucial role in teleost fish, which are lower vertebrates. Interferon (IFN) regulatory factor 5 (IRF5) is a key molecule in antiviral immunity that regulating the expression of IFN and other pro-inflammatory cytokines. It is necessary to gain more insight into the common carp IFN system and the function of fish IRF5 in the antiviral and antibacterial response. RESULTS: In the present study, we characterized the cDNA and genomic sequence of the IRF5 gene in common carp, and analysed tissue distribution and expression profile of this gene in response to polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharides (LPS) treatment. The common carp IRF5 (ccIRF5) gene is 5790 bp in length and is composed of 9 exons and 8 introns. The open reading frame (ORF) of ccIRF5 is 1554 bp, and encodes 517 amino acid protein. The putative ccIRF5 protein shares identity (65.4-90.0 %) with other fish IRF5s and contains a DNA binding domain (DBD), a middle region (MR), an IRF-associated domain (IAD), a virus activated domain (VAD) and two nuclear localization signals (NLSs) similar to those found in vertebrate IRF5. Phylogenetic analysis clustered ccIRF5 into the IRF5 subfamily with other vertebrate IRF5 and IRF6 genes. Real-time PCR analysis revealed that ccIRF5 mRNA was expressed in all examined tissues of healthy carps, with high levels observed in the gills and the brain. After poly I:C challenge, expression levels of ccIRF5, tumour-necrosis factor α (ccTNFα) and two IFN stimulated genes [ISGs (ccISG5 and ccPKR)] were up-regulated in seven immune-related tissues (liver, spleen, head kidney, foregut, hindgut, skin and gills). Furthermore, all four genes were up-regulated in vitro upon poly I:C and LPS challenges. CONCLUSIONS: Our findings suggest that IRF5 might play an important role in regulating the antiviral and antibacterial response in fish. These results could provide a clue for preventing common carp infection by pathogenic microorganisms present in the aquatic environment.

The role of Exo70 in vascular smooth muscle cell migration.

BACKGROUND: As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70's function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5. METHODS: Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay. RESULTS: The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration. CONCLUSIONS: This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty.

Knockdown of filaggrin influences the epidermal terminal differentiation via MAPK pathway in normal human epidermal keratinocytes.

We aimed to gain further insight into the role of the MAPK signaling pathway in terminal differentiation of normal human epidermal keratinocytes (NHEKs) with filaggrin knockdown. Filaggrin expression was knocked down by shRNA technology and the MAPK pathways were inhibited by three different inhibitors in NHEKs. The associated mRNAs and proteins were investigated by RT-PCR and western blot. Filaggrin absence inhibited the expression of differentiation-associated proteins, and blocked the protein expression of p38 MAPK, ERK1/2, JNK, Akt and NF-κB. Moreover, inhibited p38 MAPK, instead of ERK1/2 or JNK, lead to decreases in the expressions of Akt, NF-κB, and differentiation- associated proteins. In conclusion, Filaggrin might affect the epidermal terminal differentiation mainly through the p38-MAPK, NF-κB and Akt pathways. ERK1/2 and JNK might also be involved in the process.

Low-density lipoprotein receptor affects the fertility of female mice.

Low-density lipoprotein receptor (LDLR) has been demonstrated to play a central role in lipoprotein metabolism, with Ldlr-deficient (Ldlr(-/-)) mice developing severe dyslipidemia. In the present study we investigated whether Ldlr knockout could harm female reproduction and explored the mechanisms involved. The results indicate that although the number of litters born to Ldlr(-/-) mice did not differ significantly from that born to controls, the number of pups per litter was significantly lower in the former group. Interestingly, although Ldlr(-/-) mice were obese, the weight of their ovaries was lower than that in control mice. Serum cholesterol levels was significantly higher in Ldlr(-/-) mice than in their wild-type counterparts. In contrast, there were significant decreases in cholesterol, triglyceride and total lipid levels in ovaries of Ldlr(-/-) mice. Both ovarian lipid deposition, as detected by Oil red O staining, and lipid droplets, as evaluated by transmission electron microscopy, supported decreased lipid levels in ovaries from Ldlr(-/-) mice. In addition, Ldlr(-/-) mice had fewer ovarian follicles, more atretic follicles, lower oestrogen levels and spent significantly less time in oestrus than did the controls. Superovulation assays indicated immature Ldlr(-/-) mice ovulated fewer ova than controls. These results indicate that lack of Ldlr results in dyslipidaemia and poor fertility.

Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome.

Microsporum canis is a pathogenic fungus with worldwide distribution that causes tinea capitis in animals and humans. M. canis also causes invasive infection in immunocompromised patients. To defy pathogenic fungal infection, the host innate immune system is the first line of defense. As an important arm of innate immunity, the inflammasomes are intracellular multiprotein complexes that control the activation of caspase-1, which cleaves proinflammatory cytokine pro-interleukin-1ß (IL-1ß) into its mature form. To determine whether the inflammasome is involved in the host defense against M. canis infection, we challenged human monocytic THP-1 cells and mouse dendritic cells with a clinical strain of M. canis isolated from patients with tinea capitis. We found that M. canis infection triggered rapid secretion of IL-1ß from both THP-1 cells and mouse dendritic cells. Moreover, by using gene-specific shRNA and competitive inhibitors, we determined that M. canis-induced IL-1ß secretion was dependent on NLRP3. The pathways proposed for NLRP3 inflammasome activation, namely, cathepsin B activity, K(+) efflux, and reactive oxygen species production, were all required for the inflammasome activation triggered by M. canis. Meanwhile, Syk, Dectin-1, and Card9 were found to be involved in M. canis-induced IL-1ß secretion via regulation of pro-IL-1ß transcription. More importantly, our data revealed that M. canis-induced production of IL-1ß was dependent on the NLRP3 inflammasome in vivo. Together, this study unveils that the NLRP3 inflammasome exerts a critical role in host innate immune responses against M. canis infection, and our data suggest that diseases that result from M. canis infection might be controlled by regulating the activation of inflammasomes.

Molecular characterization of LEAP-2 cDNA in common carp (Cyprinus carpio L.) and the differential expression upon a Vibrio anguillarum stimulus; indications for a significant immune role in skin.

LEAP-2 is a cysteine-rich cationic antimicrobial peptide (AMP) playing an important role in host innate immune system. LEAP-2 genes have been identified from higher vertebrates and several fish species. Here we report the cloning and identification of two LEAP-2 cDNA sequences from the liver of common carp (Cyprinus carpio L.). The LEAP-2A cDNA was 1325 bp long and contained an ORF of 279 bp encoding a protein of 92 amino acids. The LEAP-2B cDNA was 608 bp long and contained an ORF of 276 bp encoding a protein of 91 amino acids. Both LEAP-2 proteins consisted of 41 amino acid residues and shared four cysteines at the conserved positions in the predicted mature peptides, highly similar to LEAP-2 of other species. Sequence alignment showed that LEAP-2 amino acid sequences were well conserved in different species, and the phylogenetic relation of LEAP-2 was coincident with evolution of biological species. Expression analysis data revealed that LEAP-2A and LEAP-2B mRNAs were expressed in a wide range of common carp tissues including liver, spleen, head kidney, skin, gills, hindgut and foregut. When injected intraperitoneally with Vibrio anguillarum, the expression level of common carp LEAP-2A was quickly up-regulated in liver, spleen, head kidney, skin, gills, foregut and hindgut, however, the expression level of LEAP-2B was similarly up-regulated in spleen, skin, gills and hindgut but not in liver, head kidney and foregut. Our results showed that the LEAP-2A had a markedly high constitutive expression in skin, and the LEAP-2A and the LEAP-2B had a significantly high up-regulated expression after stimulus in skin. This differential expression of LEAP-2 in common carp suggests that it may play a key role in immune responses against invading pathogens and both LEAP-2 molecules may be involved in mucosal immunity.