A Shrimp C-type Lectin Inhibits Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial Peptides

Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.     

虾类血细胞的分类与功能研究进展

血细胞在动物的免疫防御体系中扮演了重要的角色,尤其是对缺少适应性免疫的无脊椎动物.在这些动物中,血细胞既参与细胞免疫的吞噬、包囊、结节等作用,还参与体液免疫中许多免疫因子的生成与储存.对不同动物类群的血细胞的不同亚群进行区分,是深入了解其免疫机制与血细胞功能的基础.尽管国内外学者利用不同的方法,针对虾类血细胞的不同亚群...     

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.     

昆虫天然免疫相关基因研究进展

在长期进化过程中,昆虫形成了强大的天然免疫防御系统,即体液免疫和细胞免疫。体液免疫主要包括Toll、IMD和JAK/STAT 3条信号通路,通过信号转导及免疫途径调控免疫相关基因的表达,诱导产生抗菌肽和其他效应分子。细胞免疫由血细胞介导,主要完成对病原物的包裹、吞噬和集结等。近年来,昆虫基因组学快速发展,通过生物信息学等方法从昆虫基因组数据中已鉴定到大量免疫相关基因,对这些基因的研究加深了人们对昆虫天然免疫分子机制的认识和理解。根据基因功能,免疫相关基因分为识别、信号转导、调制器、效应分子、黑化反应、RNA干扰和其他基因等7类,这些基因通过互作来调控体液免疫和细胞免疫。本文对昆虫免疫相关基因的分类、功能及家族进化等方面的研究成果进行总结,并对今后昆虫免疫的研究重点进行了展望,以期为昆虫免疫分子机制的研究及开发新的害虫防治策略提供依据。     

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemo^less) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.     

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.     

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.     

Opsonic involvement in phagocytosis by mollusk hemocytes.

Macrophages from the gastrophod mollusk Otala lactea are capable of in vitro recognition and phagocytosis of foreign particles such as yeast, mammalian erythrocytes, and bacteria. The degree of intensity of the phagocytic response, in certain instances, is governed by the surface characteristics of the particle in question as well as by the presence of opsonic factors.Hemagglutinins have been implicated as opsonins in certain invertebrates, including mollusks. Otala lacks serum lectins; however, its hemolymph stimulates phagocytosis of formalized yeast but not erythrocytes and bacteria. Hemagglutinin-containing extracts of Otala albumin gland were shown to opsonize formalized red cells. The rate of ingestion of the bacteria used in this study by Otala hemocytes was variable and was not influenced by the presence of hemolymph in the medium.     

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect''s open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.     

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 μg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 μg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.     

Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis

Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

Phagocytosis as a cellular immune response mechanism in the American lobster, Homarus americanus.

Procedures to quantitate accurately the in vitro phagocytosis of sheep erythrocytes and Aerococcus viridans var. homari (formerly Gaffkya homari) bacteria by hemocytes of the American lobster were utilized to assess the effect of various vaccines on the humoral and cellular defense mechanisms of the lobster. Both the percent of hemocytes showing phagocytosis and the number of particles phagocytosed increased markedly in many of the animals injected with Pseudomonas perolens cells or endotoxin. A lesser response was elicited by A. viridans cellular vaccines. In vivo phagocytosis was observed in the circulatory system of lobsters infected with A. viridans var. homari.Natural hemolymph agglutinins for sheep or rabbit erythrocytes were not affectd by any vaccines. Hemocyte numbers dropped initially after administration of P. perolens endotoxin or cells. The bactericidin level was enhanced in samples taken from vaccinated animals.