Developmental and comparative immunology最新文献

Members of the myxovirus resistance (Mx) protein family play an essential role in antiviral immunity. They are Dynamin-like GTPases, induced by interferons. In the current study, we have characterized two predicted MX genes (MX1 and MX2) from lumpfish (Cyclopterus lumpus L.), having 12 and 13 exons, respectively. Mx2 has two isoforms (Mx2-X1 and Mx2-X2) which differ in exon 1. The lumpfish Mx proteins contain an N-terminal Dynamin-like GTPase domain, the middle domain (MD) and GTPase effector domain (GED) characteristic for Mx proteins. Phylogenetic analyses grouped all the lumpfish Mx sequences in group 1, and synteny analyses showed that both genes were localized at chromosome 5 in proximity to the genes Tohc7, Atxn7 and Psmd6. In vitro stimulation experiment showed that both MX1 and MX2-X2 were highly upregulated upon exposure to poly(I:C), but not bacteria, 24 h post exposure, indicating their role in antiviral immunity.

The interactions induced by RIP homotypic interaction motif (RHIM) are essential for the activation of inflammatory signaling and certain cell death pathways. In the present study, a RHIM-containing protein was identified from Pacific oyster Crassostrea gigas, which harbored a RHIM domain and a Death domain (designated CgRHIM-containing protein). The mRNA transcripts of CgRHIM-containing protein were constitutively expressed in all the examined tissues of oysters, with the highest expression level in mantle. The CgRHIM-containing protein was mainly distributed in the cytoplasm of oyster haemocytes. After high temperature stress, the expression levels of CgRel and CgBcl-2 increased significantly, and reached the peak level at 12 h, then decreased gradually. The transcripts of CgRHIM-containing protein, Cgcaspase-8 and Cgcaspase-3 in haemocytes up-regulated at 12 h after high temperature stress. Moreover, the protein abundance of CgRHIM-containing protein increased significantly, and the ubiquitination level of CgRHIM-containing protein in haemocytes showed an increasing trend at first and then decreased. After the expression of CgRHIM-containing protein was knocked down by siRNA, the mRNA expression levels of CgRel and CgBcl-2 decreased significantly at 6 h after high temperature stress, and those of CgFADD-like, Cgcaspase-8 and Cgcaspase-3, as well as the apoptosis rate of haemocytes also decreased significantly at 24 h. These results indicated that CgRHIM-containing protein might regulate haemocyte apoptosis in oysters upon high temperature stress via mediating the expression of Rel, Bcl-2 and caspase-8/3.

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5′-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca²⁺). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.

Stimulator of interferon genes (STING) mediates innate immune response upon binding to cyclic GMP-AMP (cGAMP). It recruits tank-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3) through its C-terminal tail and facilitates TBK1-dependent phosphorylation of IRF3 via forming STING polymers in mammalian cells. However, the mechanism behind STING-mediated activation of NF-κB transcription factor, Relish, in insect cells is unknown. Our study revealed that insect STING formed oligomers and the cryptic RIP homotypic interaction motif (cRHIM) was required for its oligomerization and its anti-viral functions. Cells expressing cRHIM-deficient mutants exhibited lower levels of anti-viral molecules, higher viral load after viral infection and weak activation of Relish. Moreover, we observed that under cGAMP stimulation, insect STING interacted with IMD, and deletion of the cRHIM motif on either protein prevented this interaction. Finally, we demonstrated that cGAMP enhanced the amyloid-like property of insect STING aggregates by ThT staining. In summary, our research showed that insect STING employed a homotypic motif to form intermolecular interactions that are essential for its antiviral signaling.

Invertebrate lectins exhibit structural diversity and play crucial roles in the innate immune responses by recognizing and eliminating pathogens. In the present study, a novel lectin containing a Gal_Lectin, a CUB and a transmembrane domain was identified from the Pacific oyster Crassostrea gigas (defined as CgGal-CUB). CgGal-CUB mRNA was detectable in all the examined tissues with the highest expression in adductor muscle (11.00-fold of that in haemocytes, p < 0.05). The expression level of CgGal-CUB mRNA in haemocytes was significantly up-regulated at 3, 24, 48 and 72 h (8.37-fold, 12.13-fold, 4.28-fold and 10.14-fold of that in the control group, respectively) after Vibrio splendidus stimulation. The recombinant CgGal-CUB (rCgGal-CUB) displayed binding capability to Mannan (MAN), peptidoglycan (PGN), D-(+)-Galactose and L-Rhamnose monohydrate, as well as Gram-negative bacteria (Escherichia coli, V. splendidus and Vibrio anguillarum), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus sybtilis) and fungus (Pichia pastoris). rCgGal-CUB was also able to agglutinate V. splendidus, and inhibit V. splendidus growth. Furthermore, rCgGal-CUB exhibited the activities of enhancing the haemocyte phagocytosis towards V. splendidus, and the phagocytosis rate of haemocytes was descended in blockage assay with CgGal-CUB antibody. These results suggested that CgGal-CUB served as a pattern recognition receptor to bind various PAMPs and bacteria, and enhanced the haemocyte phagocytosis towards V. splendidus.

Consideration is given to previous and more recent protocols for harvesting arthropod haemocytes from Galleria, Drosophila, mosquitoes, Limulus and crustaceans. The optimal harvesting of these cells is essential for meaningful studies of invertebrate immunity in vitro. The results of such experiments, however, have often been flawed due to a lack of understanding of the fragile nature of arthropod haemocytes on exposure to bacterial lipopolysaccharides, resulting in the aggregation and loss of cell types during haemolymph clotting. This article emphasizes that although there are similarities between mammalian neutrophils and arthropod haemocytes, the protocols required for the successful harvesting of these cells vary significantly. The various stages for the successful harvesting of arthropod haemocytes are described in detail and should provide invaluable advice to those requiring both high cell viability and recovery of the different cell types for subsequent experimentation.

To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is exposed to a variety of microbes due to its bacterivorous lifestyle, previous work on the components of its immune system mainly based on the description of transcriptional changes during bacterial challenges. Very few effector components of its immune system have been characterized so far. To investigate the role of lysozymes in terms of antibacterial defense and digestion, we studied a member of the widely neglected family of C. elegans invertebrate lysozymes (ILYS). We focused on the so far virtually undescribed ILYS-5, which we purified from protein extracts of C. elegans tracing its peptidoglycan-degrading activity and localized the tissue expression of the gene in vivo using a translational reporter construct. We recombinantly synthesized ILYS-5 and determined the physicochemical activity optimum and the antibacterial spectrum of a lysozyme from C. elegans for the first time. With an activity optimum at low ionic strength (≤100 mM) and at acidic pH (≤ pH 4.0), ILYS-5 is likely to be involved in killing and digestion of bacteria within acidified phagolysosomes and acidic regions of the gut, presumably secreted by lysosome-like vesicles. This notion is supported by potent activity against various live Gram-positive and Gram-negative bacteria. Notably, members of the natural associated microbiome of C. elegans are substantially less susceptible to ILYS-5. Ablation of the ilys-5 gene resulted in reduction of lifespan and fertility when cultured on the standard food bacterium Escherichia coli OP50, whereas exposure of the ilys-5 knock-out mutant to the host-associated bacterium Pseudomonas lurida MYb11 did not have a clear effect. These findings indicate a role of ILYS-5 in immunity and nutrition and a co-evolved adaptation of host and bacteria to the mutualistic nature of their interaction.

An infection induces the migration of immune cells called hemocytes to the insect heart, where they aggregate around heart valves called ostia and phagocytose pathogens in areas of high hemolymph flow. Here, we investigated whether the cardiac extracellular matrix proteins, Pericardin (Prc) and Lonely heart (Loh), regulate the infection-induced aggregation of periostial hemocytes in the mosquito, An. gambiae. We discovered that RNAi-based post-transcriptional silencing of Prc or Loh did not affect the resident population of periostial hemocytes in uninfected mosquitoes, but that knocking down these genes decreases the infection-induced migration of hemocytes to the heart. Knocking down Prc or Loh did not affect the proportional distribution of periostial hemocytes along the periostial regions. Moreover, knocking down Prc or Loh did not affect the number of sessile hemocytes outside the periostial regions, suggesting that the role of these proteins is cardiac-specific. Finally, knocking down Prc or Loh did not affect the amount of melanin at the periostial regions, or the intensity of an infection at 24 h after challenge. Overall, we demonstrate that Prc and Loh are positive regulators of the infection-induced migration of hemocytes to the heart of mosquitoes.

Infections with pathogenic Vibrio strains are associated with high summer mortalities of Pacific oysters Magalana (Crassostrea) gigas, affecting production worldwide. This raises the question of how M. gigas cultures can be protected against deadly Vibro infection. There is increasing experimental evidence of immune priming in invertebrates, where previous exposure to a low pathogen load boosts the immune response upon secondary exposure. Priming responses, however, appear to vary in their specificity across host and parasite taxa. To test priming specificity in the Vibrio – M. gigas system, we used two closely related Vibrio splendidus strains with differing degrees of virulence towards M. gigas. These V. splendidus strains were either isolated in the same location as the oysters (sympatric, opening up the potential for co-evolution) or in a different location (allopatric). We extracted cell-free haemolymph plasma from infected and control oysters to test the influence of humoral immune effectors on bacterial growth in vitro. While addition of haemolypmph plasma in general promoted growth of both strains, priming by an exposure to a sublethal dose of bacterial cells lead to inhibitory effects against a subsequent challenge with a potentially lethal dose in vitro. Inhibitory effects and immune priming was strongest when oysters had been primed with the sympatric Vibrio strain, but inhibitory effects were seen both when challenged with the sympatric as well as against allopatric V. splendidus, suggesting some degree of cross protection. The stronger immune priming against the sympatric strain suggests that priming could be more efficient against matching local strains potentially adding a component of local adaptation or co-evolution to immune priming in oysters. These in vitro results, however, were not reflected in the in vivo infection data, where we saw increased bacterial loads following an initial challenge. This discrepancy might suggests that that it is the humoral part of the oyster immune system that produces the priming effects seen in our in vitro experiments.

Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker.