The Establishment of Complement System Is from Gene Duplication and Domain Shuffling.

The mammalian complement system constitutes a highly sophisticated body defense machinery. The evolutionary origin of the complement system can be traced to Coelenterata as the presence of the central component C3 and two activation proteases BF and MASP. In the present study, the main complement components were screened and analyzed from the genomes of different species in metazoan subphyla/phyla. C1q with classical domains can be traced to Annelida, and ficolin and MBL to Urochordata. C1r and C1s are only found in Chondrichthyes and even higher species, and MASP is traced to Coelenterata. In the evolutionary tree, C1r from Vertebrates is close to MASP1/2/3 from Deuterostomia and Coelenterata, and C1s from Vertebrates is close to MASP-like protease (MASPL) from Arthropoda, Mollusca, and Annelida. C2, BF, and DF can be traced to Mollusca, Coelenterata, and Porifera, respectively. There are no clear C2 and BF branches in the evolutionary tree. C3 can be traced to Coelenterata, and C4 and C5 are only in Chondrichthyes and even higher species. There are three clear C3, C4, and C5 branches in the evolutionary tree. C6-like (C6L) and C8 can be traced to Urochordata, and C7-like (C7L) can be traced to Cephalochordara. C6L, C7L, and C8 from Urochordata and Cephalochordara provide the structural conditions for the formation of Vertebrate MAC components. The findings unveil the evolutionary principles of the complement system and provide insight into its sophistication.

A novel lectin with a distinct Gal_Lectin and CUB domain mediates haemocyte phagocytosis in oyster Crassostrea gigas.

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.

Cinobufacini enhances the therapeutic response of 5-Fluorouracil against gastric cancer by targeting cancer stem cells via AKT/GSK-3ß/ß-catenin signaling axis.

BACKGROUND: Gastric cancer stem cells (GCSCs) play crucial role in the development, recurrence, and resistance of gastric cancer (GC). Cinobufacini, a traditional Chinese medicine, offers significant advantages in improving tumor therapy. However, pre-clinical investigation into the antitumor effect and mechanism of Cinobufacini on GC is still lacking. Additionally, it has not been reported whether Cinobufacini is related to cancer stem cells (CSCs). METHODS: The CCK-8, clone formation, EdU staining, transwell and wound healing experiments were performed to assess the cell toxicity of Cinobufacini and demonstrate the preventive effects of Cinobufacini on proliferation, invasion, and migration of GC cells. Elucidating the underlying mechanism of Cinobufacini in GC based on the transcriptome sequencing. Flow cytometry assays, sphere formation assays, subcutaneous xenograft model in nude mice, and immunofluorescent staining have been used to investigate whether the anti-GC effect of Cinobufacini is associated with GCSCs and enhancing therapeutic response to 5-Fluorouracil (5-FU). RESULTS: Cinobufacini exerts minimal impact on normal human gastric epithelium cell GES-1, while significantly suppressing the proliferation, invasion, and migration of GC cell lines. Additionally, Cinobufacini attenuates the stemness of GCSCs by disrupting the AKT/GSK-3ß/ß-catenin signaling cascade. Moreover, Cinobufacin enhances the anti-tumor effects of 5-FU against GCSCs by reducing in vitro sphere formation and inhibiting subcutaneous graft tumor growth in vivo. CONCLUSIONS: Cinobufacini enhances the therapeutic response of 5-FU against GC by targeting CSCs via AKT/GSK-3ß/ß-catenin signaling axis. Our findings offer a crucial insight into the molecular mechanism of Cinobufacini's anticancer activity in GC.

ROS-responsive biomimetic nanosystem camouflaged by hybrid membranes of platelet-exosomes engineered with neuronal targeting peptide for TBI therapy.

Recovery and survival following traumatic brain injury (TBI) depends on optimal amelioration of secondary injuries at lesion site. Delivering mitochondria-protecting drugs to neurons may revive damaged neurons at sites secondarily traumatized by TBI. Pioglitazone (PGZ) is a promising candidate for TBI treatment, limited by its low brain accumulation and poor targetability to neurons. Herein, we report a ROS-responsive nanosystem, camouflaged by hybrid membranes of platelets and engineered extracellular vesicles (EVs) (C3-EPm-|TKNPs|), that can be used for targeted delivery of PGZ for TBI therapy. Inspired by intrinsic ability of macrophages for inflammatory chemotaxis, engineered M2-like macrophage-derived EVs were constructed by fusing C3 peptide to EVs membrane integrator protein, Lamp2b, to confer them with ability to target neurons in inflamed lesions. Platelets provided hybridized EPm with capabilities to target hemorrhagic area caused by trauma via surface proteins. Consequently, C3-EPm-|PGZ-TKNPs| were orientedly delivered to neurons located in the traumatized hemisphere after intravenous administration, and triggered the release of PGZ from TKNPs via oxidative stress. The current work demonstrate that C3-EPm-|TKNPs| can effectively deliver PGZ to alleviate mitochondrial damage via mitoNEET for neuroprotection, further reversing behavioral deficits in TBI mice. Our findings provide proof-of-concept evidence of C3-EPm-|TKNPs|-derived nanodrugs as potential clinical approaches against neuroinflammation-related intracranial diseases.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

A galectin-9 involved in the microbial recognition and haemocyte autophagy in the Pacific oyster Crassostrea gigas.

Galectin-9 is a tandem-repeat type member of galectin family participating in various immune responses, such as cell agglutination, phagocytosis, and autophagy. In the present study, a tandem repeat galectin-9 (defined as CgGal-9) was identified from Pacific oyster Crassostrea gigas, which consisted of two conserved carbohydrate recognition domains (CRDs) joined by a linker peptide. CgGal-9 was closely clustered with CaGal-9 from C. angulata, and they were assigned into the branch of invertebrate galectin-9s in the phylogenetic tree. The mRNA transcripts of CgGal-9 were detected in all the tested tissues, with the highest expression level in haemocytes. The mRNA expressions of CgGal-9 in haemocytes increased significantly after lipopolysaccharide (LPS) and Vibrio splendidus stimulation. The recombinant CgGal-9 was able to bind all the examined pathogen-associated molecular patterns (LPS, peptidoglycan, and mannose) and microbes (V. splendidus, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Pichia pastoris), and agglutinated most of them in the presence of Ca2+. In CgGal-9-RNAi oysters, the mRNA expressions of autophagy related genes (CgBeclin1, CgATG5, CgP62 and CgLC3) in haemocytes decreased significantly while that of CgmTOR increased significantly at 3 h after V. splendidus stimulation. The autophagy level and mRNA expressions of autophagy related genes decreased in haemocytes after CgGal-9 was blocked by the corresponding antibody. These results revealed that CgGal-9 was able to bind different microbes and might be involved in haemocyte autophagy in the immune response of oyster.

A autophagy related-like protein 16-1 promotes the formation of autophagosomes and autolysosomes in antibacterial immune response of Pacific oyster Crassostrea gigas.

Autophagy related 16-like (ATG16L) protein is a core autophagy protein, which promotes the extension of autophagosome membrane through microtubule-associated protein light chain 3 (LC3). In the present study, an ATG16L was identified from oyster Crassostrea gigas (defined as CgATG16L1). The full-length cDNA of CgATG16L1 was of 3184 bp with an open reading frame of 1650 bp that encoded a polypeptide of 549 amino acids. There was an ATG5-interacting motif (AFIM) domain, a coiled-coil (CC) domain and seven tryptophan-aspartic acid 40 (WD40) repeats in CgATG16L1. ATG16L1 mRNA was expressed in all the examined tissues with the highest expression in haemolymph (11.22-fold of that in hepatopancreas, p < 0.05). The mRNA expressions of CgATG16L1 in haemocytes increased significantly at 3, 6, 12, 24 and 72 h after lipopolysaccharide (LPS) stimulation, which were 81.15-fold, 24.95-fold, 6.02-fold, 3.90-fold and 5.97-fold (p < 0.05) of that in control group, respectively. The green positive signals of CgATG16L1 protein and the red positive signals of CgLC3 protein were dotted in the cytoplasm of agranulocytes, semi-granulocytes and granulocytes. The co-localization of CgATG16L1 and CgLC3 was observed in haemocytes after Vibrio splendidus stimulation. In CgATG16L1-RNAi oysters, the number of autophagosomes and autolysosomes in haemocytes was reduced. All these results suggested that CgATG16L1 participated in the bacteria-induced autophagy process in the haemocytes of oyster response to bacteria invasion.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

Syk regulates the haemocyte autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 in oyster antibacterial immunity.

Spleen tyrosine kinase (Syk) is reported to be involved in activating the autophagy. Recently, a homologue of Syk was identified from Pacific oyster Crassostrea gigas (defined as CgSyk). In the present study, the molecular characteristics of CgSyk and its regulation mechanism in autophagy were investigated in oyster C. gigas. The full-length cDNA of CgSyk was of 4566 bp with an open reading frame (ORF) of 1989 bp. CgSyk encoded a polypeptide of 662 amino acids, containing two Src homology 2 (SH2) domains and one tyrosine kinase catalytic (TyrKc) domain. The deduced amino acid sequence of CgSyk shared low similarity with the previously identified Syks from other species. In the phylogenetic tree, CgSyk was first clustered with Crassostrea virginica CvSyk, and then classified into a branch of invertebrate Syks. In CgSyk-RNAi oysters, the mRNA expressions of CgLC3, CgP62, CgBeclin-1 and CgATG5 in haemocytes decreased significantly at 12 h after Vibrio splendidus stimulation. At the same time, the abundance of CgLC3Ⅱ in haemocytes, and the autophagy rate of haemocytes in CgSyk-RNAi oysters decreased significantly at 12 h after V. splendidus stimulation. All the results collectively suggested that CgSyk regulated the autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 to defend against bacterial invasion in oysters.

An immunoglobulin superfamily member (CgIgIT2) functions as immune inhibitory receptor to inhibit the inflammatory cytokine expressions in Crassostrea gigas.

Immune inhibitory receptors are increasingly acknowledged as potent regulators of immune response, which inhibit the overactivation of immune system and play an important role in maintaining immune homeostasis. In the present study, a novel immunoglobulin superfamily member (CgIgIT2) was identified from the Pacific oyster, Crassostrea gigas. The protein sequence of CgIgIT2 contained one signal peptide, four Ig domains, one fibronectin type III domain, one transmembrane domain, and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM). The mRNA transcripts of CgIgIT2 were widely expressed in all the tested tissues, including haemolymph, gill, mantle, adductor muscle, labial palp, gonad and hepatopancreas, with the highest expression in haemolymph. The mRNA expressions of CgIgIT2 in haemocytes increased significantly at 24, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgIgIT2 protein were mainly detected in granulocytes of haemocytes, which were 1.27-fold and 2.15-fold (p < 0.05) higher than that of semi-granulocytes and agranulocytes, respectively. And CgIgIT2 was mainly located in the membrane and cytoplasm of haemocytes. The recombinant protein of CgIgIT2-4 × Ig (rCgIgIT2-4 × Ig) exhibited binding activity towards multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN), mannose (MAN) and polyinosinic-polycytidylic acid (Poly (I: C)) with the highest affinity for LPS. rCgIgIT2-4 × Ig could also bind Gram-negative bacteria (V. splendidus, V. anguillarum, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Pichia pastoris). In the blocking assay with anti-CgIgIT2 antibody, the mRNA expressions of interleukins (CgIL17-1, CgIL17-3 and CgIL17-6) and tumor necrosis factors (CgTNF-1 and CgTNF-2) in haemocytes all increased significantly at 12 h after V. splendidus stimulation. These results suggested that CgIgIT2 could function as an inhibitor receptor to bind different PAMPs and microbes, as well as inhibit the mRNA expressions of multiple inflammatory cytokines in oysters.

The ferroptosis in haemocytes of Pacific oyster Crassostrea gigas upon erastin treatment.

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA_permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes.

CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

The primitive complement system in molluscs.

The complement system is an important immune defense mechanism that plays essential roles in both innate and adaptive immunity of vertebrates. Since complement components are identified in deuterostome and even primitive protostome species, the origin and evolution of complement system in invertebrates have been of great interest. Recently, research on the complement system in mollusc immunity has been increasing due to their importance in worldwide aquaculture, and their phylogenetic position. Complement components including C3, C1q domain containing protein (C1qDCP), C-type lectin (CTL), ficolin-like, mannose-binding lectin (MBL)-associated serine proteases like (MASPL), and factor B have been identified, suggesting the existence of complement system in molluscs. The lectin pathway has been outlined in molluscs, which is initiated by CTL with CCP domain and MASPL protein to generate C3 cleavage fragments. The molluscan C1qDCP exhibits the capability to bind human IgG, indicating the existence of possible C1qDCP-mediated activation pathway in molluscs. The activation of C3 regulates the expressions of immune effectors (cytokines and antibacterial peptides), mediates the haemocyte phagocytosis, and inhibits the bacterial growth. Some MACPF domain containing proteins may replace the missing terminal pathway in molluscs. This article provides a review of complement system in molluscs, including its components, activation mechanisms and functions in the immune response of molluscs.

CgTNF-2 promotes the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in the Pacific oyster Crassostrea gigas.

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU+ cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI+ haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas.

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

Lung-Targeted Delivery of Cepharanthine by an Erythrocyte-Anchoring Strategy for the Treatment of Acute Lung Injury.

As one of the most frequent complications of critical illness, acute lung injury (ALI) carries a high risk of clinical morbidity and mortality. Cepharanthine (CPA) has significant anti-inflammatory activity, however, due to poor water solubility, low bioavailability, and short half-life, it fails to provide effective clinical management measures. Here, we explored the flexibility of an erythrocyte-anchoring strategy using CPA-encapsulated chitosan-coating nanoparticles (CPA-CNPs) anchored onto circulating erythrocytes for the treatment of ALI. CPA-CNPs adhered to erythrocytes successfully (E-CPA-CNPs) and exhibited high erythrocyte adhesion efficiency (>80%). Limited toxicity and favorable biocompatibility enabled further application of E-CPA-CNPs. Next, the reticuloendothelial system evasion features were analyzed in RAW264.7 macrophages and Sprague-Dawley rats. Compared with bare CPA-CNPs, erythrocyte-anchored CNPs significantly decreased cellular uptake in immune cells and prolonged circulation time in vivo. Notably, the erythrocyte-anchoring strategy enabled CNPs to be delivered and accumulated in the lungs (up to 6-fold). In the ALI mouse model, E-CPA-CNPs attenuated the progression of ALI by inhibiting inflammatory responses. Overall, our results demonstrate the outstanding advantages of erythrocyte-anchored CPA-CNPs in improving the pharmacokinetics and bioavailability of CPA, which offers great promise for a lung-targeted drug delivery system for the effective treatment of ALI.