Fish and shellfish immunology reports最新文献

Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a translation repressor downstream of mTORC1 pathway, and plays a pivotal role in the adaptive immune response. However, whether 4E-BP1 participates in the regulation of adaptive immunity of early vertebrates is still unclear. In present study, using Nile tilapia Oreochromis niloticus as a model, we investigated the regulatory roles of 4E-BP1 (On-4E-BP1) on lymphocyte-mediated adaptive immune response of fish species. On-4E-BP1 is highly conserved compared with the homologues from other vertebrates, and it is widely distributed in the immune-related tissues of Nile tilapia with the highest expression level in the gill. Once the animals were infected by Aeromonas hydrophila, transcription level of On-4E-BP1 in spleen lymphocytes was significantly induced on day 5 after infection. Meanwhile, phosphorylation of On-4E-BP1 in lymphocytes was also enhanced during the primary adaptive immune response, suggesting that 4E-BP1 is involved in the adaptive immunity of Nile tilapia. Furthermore, when lymphocytes were activated by agonist PMA or T cell specific mitogen PHA in vitro, phosphorylation of On-4E-BP1 was obviously up-regulated. More importantly, once mTORC1/4E-BP1 activity was blocked by specific inhibitor, the inducible expression of T cell activation markers IFN-γ and CD122 was severely impaired during PHA-induced T cell activation, suggesting this signaling regulates T lymphocyte activation of Nile tilapia. In addition, blockade of mTORC1/4E-BP1 axis also resulted in a crippled proliferation of lymphocyte during the primary response of anti-bacterial adaptive immunity. Collectively, we found that mTORC1/4E-BP1 signaling participates in the adaptive immune response by regulating lymphocyte activation and proliferation in Nile tilapia. This study enriches our current knowledge on teleost adaptive immunity, and provides novel perspective to understand the evolution of adaptive immune system.

Poly(I:C) is a kind of chemosynthetic double-stranded RNA (dsRNA) analogue which could act as TLR3 agonist and induce IFN production. It is widely applied in anti-virus treatment and immunoregulation, as well as vaccine adjuvant in farmed animals. However, whether poly(I:C) could activate innate immune response to defense against bacterial infection remains unclear. In this study, we established a feeding trial model with different dose of poly(I:C) in turbot larvae, then challenged with Edwardsiella piscicida after 3–7 weeks resting period. The results show that feeding turbot with poly(I:C) exhibited a stronger inflammatory response and antioxidant stress ability, and significantly elevated the survival rate within the decreased bacterial loads. Importantly, the bacterial infection-induced white feces in hindgut of turbot were significantly alleviated after poly(I:C) feeding, and this administration induced protection could last for about 7 weeks. Taken together, these findings indicate that feeding turbot with poly(I:C) could enhance a long-term intestinal mucosal immunity in response to bacterial infection, suggesting that poly(I:C) might be a promising immunostimulant in aquaculture.

Dendritic cells (DCs) are the professional antigen presenting cells, which play important roles in regulating the immune response and tolerance. In mammals, CD83 is considered as a maturation marker for DCs, which is a type I membrane glycoprotein. In this research, the gene sequence of the CD83 in Paralichthys olivaceus was obtained on NCBI, the sequence characteristics were studied and CD83 was detected in the head kidney, spleen, gut, gill, liver and muscle, with lower expression in the liver and muscle and higher expression in the head kidney and spleen. And then the CD83 recombinant protein was expressed and the specific antibodies (Abs) were prepared, transiently expressed CD83 eukaryotic proteins could be identified by western blot and indirect immunofluorescence. Then single cell suspensions of head kidney were obtained and DCs were collected by density gradient centrifugation after 7 days of cultivation. Giemsa staining revealed that the nuclei were kidney and dumbbell shaped, and the cell surfaces had dendritic or pseudopod-like protrusions. In addition, the DCs could phagocytose fluorescent microspheres, and the phagocytosis rate was 79.27±1.01%. After incubation with allogeneic lymphocytes, it was found that the lymphocytes clustered and proliferated significantly with the ratio ranging from 39.0% to 59.8% in a dose-dependent manner, which proved that the DCs could elicit mixed lymphocyte responses. After LPS immunization in vivo, the relative expression of CD83 in the head kidney and spleen showed a trend of increasing and then decreasing, peaking at 6 h. Meanwhile, the expression of CD83, MHC II, CD80/86 and CD40 were significantly up-regulated in DCs after stimulated with LPS for 24 h in vitro. And then IIF revealed that CD83⁺ cells could be detected in the LPS-stimulated group using CD83 Abs, while no positive signal was detected in the control group, which suggested that CD83 protein may be considered as a specific marker for the maturation of DCs in teleost.

Gill diseases may cause high mortalities in farmed Atlantic salmon. In seawater reared fish co-infections involving the epitheliocystis associated bacterium Ca. Branchiomonas cysticola, the microsporidian Desmozoon lepeophtherii, the causative agent of amoebic gill disease Paramoeba perurans and salmon gill poxvirus are common and histopathological lesions may be complex. Here, we report detection of these agents utilising multiplex real-time PCR and link the presence of agents to histopathologically visible gill lesions by in situ hybridisation (ISH) utilising RNAscope®. We show that Ca. Branchiomonas cysticola infections may remain undetected if diagnostic investigations are restricted to histopathology alone. Further, positive in situ labelling of Ca. Branchiomonas cysticola was observed within epitheliocysts, but also in small foci within areas of inflammation and necrosis in which histologically detectable epitheliocysts were not visible. In situ labelling of D. lepeophtherii corresponded well with tissue distribution patterns previously associated with this microsporidian. Salmon gill poxvirus was associated with apoptotic gill epithelial cells, while Ca. Piscichlamydia salmonis could not be associated with pathological changes. The multiplex real-time PCRs utilised were rapid and sensitive diagnostic tools and the results corresponded well with ISH. This study shows that the agents involved in complex gill disease can be linked to lesions using ISH and suggests that Ca. B. cysticola plays a crucial role in the development of gill disease in the farming of salmon in Norway.

Mast cells are important in inflammatory processes and in the nonspecific immune response, and there are also indications that these cells are associated with the effectors of the specific immune response. Eosinophilic granular cells are frequently compared to mast cells, and some authors maintain that they are the same cells. In this study, we take a fresh look at the similarities and differences between these two cell types in Oncorhynchus mykiss. We evaluated the cytomorphology of each cell type with optical microscopy, their staining affinities, and their ultrastructure. We observed that mast cells were positive for CD117 (c-kit), while eosinophilic granular cells were negative for this marker. We propose that these two cell types have certain common characteristics but represent well-differentiated populations distributed in several Oncorhynchus mykiss tissues.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) sense microbial-associated molecular patterns (MAMPs) to activate the innate immune responses. RLRs in aquatic animals include RIG-I, MDA5, LGP2, mollusc RIG-I-like and MDA5-like proteins, which exhibit structural and functional diversity. RLRs data from 30 species of aquatic animals were collected for analysis. Not all species contain all the RLR members. RIG-I is absent in the orders of Perciformes, Cichliformes and Pleuronectiformes, and LGP2-like protein is absent in the class of Bivalvia. Due to the differences in species and variants, the homologous proteins have different responses to pathogens. LGP2 works as an immune homeostasis regulator to balance the immune response. The phylogenetic analysis demonstrates RIG-I and mollusc RIG-I-like present in the same evolutionary branch and appear in the early stage. RIG-I is an ancient PRR in innate antiviral immunity. The prototype of antiviral immune system has emerged in aquatic mollusc. Aquatic animal RLRs were analyzed and summarized in terms of structures, functions, and evolutions, which provide the basic information for future researches in RLRs. Base on the diversity of species and RLRs, more and in-depth studies in aquatic animal RLRs should be done.

Penaeidins are members of an antimicrobial peptide (AMP) family that have broad anti-microbial activities only found in penaeid shrimps. The LvBigPEN, a member of penaeidins from shrimp Litopenaeus vannamei, has showed antiviral activity against white spot syndrome virus (WSSV) in our previous report. However, whether LvBigPEN possesses potential anti-bacterial activities is still unknown. Herein, we found that the LvBigPEN played an important role in restricting the infection of Vibrio parahaemolyticus, a natural and Gram-negative bacteria pathogen in shrimp. The transcription of LvBigPEN was strongly induced after V. parahaemolyticus challenge. RNA interference (RNAi) mediated knockdown of LvBigPEN showed that LvBigPEN had a potential antibacterial function against V. parahaemolyticus. Microorganism binding assays indicated that rLvBigPEN could bind to both Gram-negative bacteria and Gram-positive bacteria. Transmission electron microscopy (TEM) analysis showed its ability to destroy bacterial cells in vitro. Besides, in a gel retardation assay, rLvBigPEN could bind to plasmid DNA and bacteria (V. parahaemolyticus) genomic DNA in a concentration-dependent manner. Moreover, the AP-1 pathway could participate in the transcription of LvBigPEN by the dual luciferase reporter assays. Taken together, these results suggested that LvBigPEN possessed the antibacterial activity against V. parahaemolyticus and may be alternative agents for the prevention and treatment of diseases caused by V. parahaemolyticus.

The study is aimed to investigate the protective effect and potential mechanisms of sodium butyrate (NaBT) on soyasaponins (SA) induced intestinal epithelial cells (IECs) injury in vitro. The primary IECs of turbot were developed and treated with 0.4, 1 and 4 mM NaBT in the presence of 0.4 mg/mL SA for 6 h to explore the protective effects of NaBT. The results showed that the addition of NaBT significantly down-regulated gene expression of inflammatory cytokine TNF-α, IL-1β and IL-8, pro-apoptosis relevant gene BAX, caspase-3, caspase-7 and caspase-9 induced by SA, while up-regulated anti-apoptosis gene Bcl-2. SA stimulation did not induce reactive oxygen species production, but elevated gene expression of antioxidant enzyme heme oxygenase-1 and superoxide dismutase. Moreover, the gene expression of those antioxidant enzyme was further up-regulated in NaBT groups. Furthermore, NaBT supplementation decreased the acid phosphatase and alkaline phosphatase activities and suppressed phosphorylation of p38 and c-Jun N-terminal kinase (JNK). In conclusion, NaBT could mitigate SA-induced inflammation and apoptosis and elevate gene expression of antioxidant enzymes on IECs of turbot and p38 and JNK signaling pathway participated in those processes.

Mitochondria are organelles commonly associated with adenosine triphosphate (ATP) formation through the oxidative phosphorylation (OXPHOS) process. However, mitochondria are also responsible for functions such as calcium homeostasis, apoptosis, autophagy, and production of reactive oxygen species (ROS) that, in conjunction, can lead to different cell fate decisions. Mitochondrial morphology changes rely on nutrients’ availability and the bioenergetics demands of the cells, in a process known as mitochondrial dynamics, which includes both fusion and fission. This organelle senses the microenvironment and can modify the cells to either a pro or anti-inflammatory profile. The zebrafish has been increasingly used to research mitochondrial dynamics and its connection with the immune system since the pathways and molecules involved in these processes are conserved on this fish. Several genetic tools and technologies are currently available to analyze the behavior of mitochondria in zebrafish. However, even though zebrafish presents several similar processes known in mammals, the effect of the mitochondria in the immune system has not been so broadly studied in this model. In this review, we summarize the current knowledge in zebrafish studies regarding mitochondrial function and immuno metabolism.

Calnexin (CNX) is one of the major calcium-binding proteins in endoplasmic reticulum (ER) and plays a crucial role in regulating Ca²⁺ homeostasis and unfolded protein response (UPR). In the present study, PyCNX was identified in Yesso Scallop Patinopecten yessoensis. The open reading frame (ORF) of PyCNX was of 1794 bp encoding a putative polypeptide of 598 amino acid residues with an N-terminal signal peptide, a typical calreticulin (CRT) domain and a C-terminal transmembrane domain. The deduced amino acid sequence of PyCNX shared 47.91%-70.16% identities with CNXs from other species. The mRNA transcripts of PyCNX were constitutively expressed in all the examined tissues, including gills, gonad, hepatopancreas, mantle and haemocytes, with the highest expression level in gills. The mRNA transcripts of PyCNX in gills were significantly up-regulated at 1 h (p < 0.01), down-regulated to similar level of Blank group at 3 h (p > 0.05) and 6 h (p > 0.05), and decreased significantly from 12 to 48 h (p < 0.05) after acute high temperature stress (25 °C). PyCNX was mainly located in the ER of haemocytes. The expression profiles of PyATF6, PyIRE1 and PyGRP78 in the gills of scallops after acute high temperature stress were investigated using quantitative real-time PCR. The mRNA transcripts of PyATF6 increased significantly at 1 h, 3 h and 6 h after acute high temperature stress (p < 0.01), then down-regulated to similar level of Blank group at 12 h (p > 0.05) and 24 h (p > 0.05), and finally significantly up-regulated again at 48 h (p < 0.05). Similar to PyATF6, the mRNA transcripts of PyIRE1 were significantly up-regulated at 1 h (p < 0.05), 3 h (p < 0.01), 6 h (p < 0.05) and 48 h (p < 0.05) after acute high temperature stress. The mRNA transcripts of PyGRP78 were significantly up-regulated at 3 h (p < 0.05), reached the highest level at 6 h (p < 0.01) after acute high temperature stress, and kept significant higher level at 12-48 h (p < 0.05). These results indicated that PyCNX was involved in the response upon the acute high temperature stress of scallops probably by regulating UPR.