Cellular encapsulation and melanization are enhanced by immulectins, pattern recognition receptors from the tobacco hornworm Manduca sexta

An effective innate immune response against parasites in insects is encapsulation followed by melanization. In cellular encapsulation, formation of capsules requires plasmatocytes and granulocytes, the two most abundant hemocytes in lepidopteran insects. We have developed an in vitro encapsulation assay to elucidate functions of immulectins in encapsulation. Immulectins are members of the C-type (calcium-dependent) lectin superfamily, and they function as humoral pattern recognition receptors in innate immunity of the tobacco hornworm Manduca sexta. We demonstrate that coating of immulectins to agarose beads enhanced cellular encapsulation in vitro. Immulectin-1 enhanced encapsulation but not melanization. Encapsulation occurred with isolated hemocytes that lack plasmatocytes when agarose beads were coated with immulectins. However, plasmatocytes were required for aggregation of encapsulated beads. We also showed that inhibitors of serine proteinases such as para-aminophenylmethylsulfonyl fluoride (p-APMSF) inhibited cellular encapsulation of immulectin-coated agarose beads. Our results suggest that humoral pattern recognition receptors may enhance cellular encapsulation and melanization.     

Immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, Manduca sexta

In insects, encapsulation followed by melanization is a major defense mechanism against metazoan parasites. However, insects must recognize and differentiate nonself before they mount an immune response. Recognition of pathogens in insects is accomplished by a set of pattern recognition receptors (PRRs). Binding of PRRs to pathogens is linked to a variety of immune responses including phagocytosis, nodule formation, encapsulation, and prophenoloxidase activation. So far, little is known about how recognition of pathogens by PRRs triggers different immune responses. In this article, we report that immulectin-2, a C-type lectin, enhances encapsulation and melanization processes in the tobacco hornworm, Manduca sexta. Coating of agarose beads with recombinant carboxyl-terminal carbohydrate-recognition domain (CRD2-II) of immulectin-2 enhanced encapsulation of the beads in vitro by hemocytes and melanization of the beads in vivo in M. sexta larvae. Recombinant CRD2-II also directly bound to granular cells and oenocytoids, but not to plasmatocytes or spherule cells. Immulectin-2 in hemolymph of M. sexta larvae bound to the surface of a nematode, Caenorhabditis elegans, and recombinant CRD2-II directly bound to C. elegans and a human filarial nematode, Brugia malayi. Binding of CRD2-II to C. elegans enhanced melanization of the nematode in vivo. Our results suggest that binding of immulectin-2 to the surface of parasites can trigger encapsulation and melanization responses in M. sexta.     

Involvement of a pattern recognition receptor C-type lectin 7 in enhancing cellular encapsulation and melanization due to its carboxyl-terminal CRD domain in the cotton bollworm, Helicoverpa armigera

C-type lectins play important roles in innate immunity as pattern recognition receptors (PRRs). We have previously reported a novel C-type lectin HaCTL7 from the cotton bollworm (Helicoverpa armigera) which contains two carbohydrate-recognition domains (CRDs), namely N-terminal CRD1 and C-terminal CRD2. Interestingly, there are four but not six of conserved cysteine residues in CRD2 of HaCTL7, which is different from that of other dual CRD C-type lectins. In the current study, we expressed and purified recombinant HaCTL7 (rHaCTL7) as well as rCRD1 and rCRD2, and demonstrated that both rHaCTL7 and rCRD2, but not rCRD1, owned the agglutinate ability against both Gram-negative and Gram-positive bacteria in a calcium dependent manner. In addition, both rHaCTL7 and rCRD2, but not rCRD1, could bind to various bacteria, and enhanced haemocytes mediated encapsulation and melanization processes. HaCTL7 secreted from fat bodies is able to bind to granulocytes, plasmatocytes and oenocytoids, but not to spherulocytes. Recombinant HaCTL7 and rCRD2 are capable of binding to both granulocytes and oenocytoids, while rCRD1 can only bind to granulocytes. Our data suggest that as a PRR HaCTL7 enhances encapsulation and melanization likely through its C-terminal CRD2, but not N-terminal CRD1, which imply that the characteristic four cysteine structure of CRD2 plays key roles in innate immunity.     

Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity

In innate immunity, pattern recognition receptors discriminate between self- and infectious non-self-matter. Mammalian homologs of the Drosophila Toll protein, which are collectively referred to as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS) and lipoproteins, whereas the Drosophila Toll protein does not act as a PAMP receptor, but rather binds to Sp?tzle, an endogenous peptide. In Drosophila, innate immune surveillance is mediated by members of the peptidoglycan recognition protein (PGRP) family, which recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions including the release of antimicrobial peptides and the activation of the prophenoloxidase cascade, the latter effecting localized wound healing, melanization, and microbial phagocytosis. In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte.     

Cellular-mediated reactions to foreign organisms inoculated into the hemocoel of Anopheles albimanus (Diptera: Culicidae)

The immune response against different organisms and particles inoculated in the hemocoel of female Anopheles albimanus Wiedemann was investigated. Histological and ultrastructural observations indicated that melanization and hemocyte type participation varied according to the particles inoculated. The initial responses against heat-killed Microccocus lysodeikticus and Escherichia coli included hemocyte lysis and melanization whereas the response to heat-killed Saccharomyces cerevisiae was only cellular, and an initial melanization of Sephadex G-25 (neutral charged) beads was followed by the formation of cellular aggregates. After 24 h, hemocytes were involved in all terminal encapsulation events. Plasmodium vivax Grassi and Feletti formalin-fixed sporozoites induced a weak response. Cellular aggregates were observed 1 h postinoculation, but participating hemocytes could not be identified because of the extensive cellular damage and lysis. Sporozoites were also observed in the core of these aggregates, mixed with cell debris and free in the hemolymph. The effect on the inoculated particles was also different-S. cerevisiae was encapsulated only by hemocytes, whereas M. lysodeikticus was lysed and E. coli was phagocytosed by plasmatocytes. These results indicate that hemocytes are important components in the immune response in An. albimanus.     

Identification and characterization of a QM protein as a possible peptidoglycan recognition protein (PGRP) from the giant tiger shrimp Penaeus monodon

In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (accession numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2 h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes.     

Mechanism by which Bombyx mori hemocytes recognize microorganisms: direct and indirect recognition systems for PAMPs

Hemocytes play an important role in cellular reactions in the immune system. Although the recognition of pathogens is thought to involve pattern-recognition proteins (PRPs) in insects, the exact mechanisms by which insect hemocytes recognize pathogens are not clear. This study examined the mechanism by which Bombyx mori hemocytes recognize microorganisms and pathogen-associated molecular patterns (PAMPs) using flow cytometry and fluorescence microscopy. Fluorescence-labeled bacterial or fungal cells were observed to bind to hemocytes and this binding was inhibited by adding lipoteichoic acid (LTA) or beta-1,3-glucan. Lipopolysaccharide (LPS) bound to hemocytes directly. These results suggest that hemocytes have a mechanism that recognizes LPS, LTA, and beta-1,3-glucan directly. Previously, we identified two types of C-type lectin (BmLBP and BmMBP) and showed that they recognize a variety of PAMPs leading to the induction of nodule formation. These lectins enhanced hemocyte binding to microorganisms and their direct binding to hemocytes suggests that hemocytes have a mechanism for recognizing microorganisms using lectin receptors.     

Ostrinia furnacalis integrin β1 may be involved in polymerization of actin to modulate spreading and encapsulation of plasmatocytes

Insect hemocytes must change their state from non-adhesive to adhesive when they spread on or encapsulate foreign invaders. Although integrin β has been reported to play an important role in hemocyte spreading and encapsulation in several insects, how it is involved in the encapsulation process is still unclear. Here we report that integrin β1 of Ostrinia furnacalis (Ofint β1) may modulate plasmatocyte spreading by regulating polymerization of F-actin and further affecting formation of capsules. In the Sephadex A-25 bead-injected larvae, hemocytes forming capsules expressed approximately ten times more Ofint β1 than hemocytes that are free in circulation in hemolymph. When the expression of Ofint β1 in hemocytes was inhibited by dsRNA of Ofint β1 (dsINT), polymerization of F-actin in hemocytes, especially in plasmatocytes, was significantly decreased, spreading of plasmatocytes was inhibited, and encapsulation rate of Sephadex beads was also significantly decreased. Furthermore, hemocytes formed individual aggregates on beads in the dsINT injected larvae, while hemocytes formed complete capsules surrounding the beads in the control larvae; and most of the hemocytes on the beads in the dsINT-injected larvae assumed round forms rather than spread forms. Based on these results, we speculate that integrins on cellular membranes may modulate hemocyte spreading by regulating polymerization of F-actin and further affecting encapsulation of foreign objects.     

Expression profiles of six novel C-type lectins in response to bacterial and 20E injection in the cotton bollworm (Helicoverpa armigera)

C-type lectins can act as pattern recognition receptors (PRRs) and play an important role in innate immunity. Two C-type lectins (HaCTL1 and HaCTL2) have been previously identified in the cotton bollworm (Helicoverpa armigera). Here we isolate six C-type lectins from H. armigera (HaCTL3, 4, 5, 6, 7 and 8). All six new HaCTLs encode a signal peptide (or partial signal peptide) and complete tandem carbohydrate-recognition domains (CRDs). HaCTL4, 5, 6, 7 and 8 mRNA increased in the fat body after injection with both killed Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, whereas HaCTL3 mRNA was upregulated following E. coli injection only. Recombinant HaCTL3 exhibited agglutinating activity against both Gram-negative and Gram-positive bacteria in a calcium-dependent manner. Agglutination inhibitory analysis indicated that rHaCTL3 recognizes maltose, trehalose, peptidoglycan and lipopolysaccharides. HaCTL3 and HaCTL8 mRNA showed upregulation while HaCTL4, 5, and 6 mRNA downregulation post 20-Hydroxyecdysone (20E) injection. Our results indicate that the six novel C-type lectins of H. armigera may play important roles in defending against bacteria as PRRs and the hormone 20E can function in regulating immunity through lectins.     

Two antibacterial C-type lectins from crustacean,Eriocheir sinensis,stimulated cellular encapsulation in vitro

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate.     

Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate.     

Nuclear translocation of immulectin-3 stimulates hemocyte proliferation

Immulectin-3 (IML-3) is a C-type lectin from the tobacco hornworm Manduca sexta that contains a motif (NWGV) similar to the BH1 motif (NWGR) of the mammalian galectin-3. IML-3 is synthesized in fat body and secreted into hemolymph, but can be translocated into hemocytes. In this study, we showed that IML-3 was predominantly localized to the nucleus of hemocytes and some metaphase, anaphase and telophase hemocytes from M. sexta larvae injected with bacterial lipopolysaccharide (LPS). IML-3 was detected in the membrane and soluble extracts of hemocytes, suggesting that it may be translocated into hemocytes via receptor-mediated endocytosis. To investigate the role of IML-3 translocation to the nucleus, we expressed recombinant wild-type IML-3 and a deletion mutant DeltaIML-3 that has the NWGV motif deleted in Drosophila S2 cells. We found that recombinant wild-type IML-3, but not DeltaIML-3, was localized to the nucleus of some S2 cells and also detected in the nuclear extract. Expression of recombinant wild-type IML-3, but not DeltaIML-3 or GFP, increased the number of proliferating S2 cells. Our results suggest that nuclear translocation of IML-3 may stimulate hemocyte proliferation.     

A novel prophenoloxidase 2 exists in shrimp hemocytes

The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.     

A novel lectin domain-containing protein (LvCTLD) associated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannamei to yellow head virus (YHV)

When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system.     

Analysis of EST and lectin expressions in hemocytes of Manila clams (Ruditapes philippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni

The hemocytes of invertebrates play key roles in both cellular and humoral immune reactions by phagocytosis or delivering immune factors such as lectin and anti-microbial peptides. Bacterial infection causes changes in components such as lectins, anti-bacterial peptides, and lysosomal enzymes of plasma or hemolymph in molluscs. Previously, we found that infection with the protozoan parasite, Perkinsus, increases lectin synthesis in hemocytes. In order to investigate the patterns of genes expressed in Manila clams (Ruditapes philippinarum) infected with the protozoan parasite Perkinsus olseni, we constructed a cDNA library and sequenced 1850 clones (expressed sequence tags). A total of 79 ESTs, were related to 29 functional immune genes such as C-type lectin, lysozyme, and cystatin B, in Manila clams. Lectins were the largest group of immune-function ESTs found in our Manila clams library. Among 7 lectin clones, two full length cDNAs of lectins were cloned. MCL-3, which is a simple C-type lectin composed of 151 amino acids, has a relatively short signal sequence of 17aa and single carbohydrate-recognition domain (CRD) of 130 residues. It is highly homologous to eel C-type lectin. The sequence of mc-sialic acid-binding lectin consists of 168 amino acid residues with molecular weight of 19.2 and shows high homology to sialic acid-binding lectin from the snail, Cepaea hortensis.

The expression of 7 different lectins in hemocytes was analyzed by RT-PCR using gene-specific primers. Hemocytes from Perkinsus-infected clam expressed different sets of lectins than with Vibrio infection. These results demonstrate that several lectins are involved in Manila clam innate immunity and different challenges induce expression of different lectins.     

Identification and comparative analysis of three novel C-type lectins from the silkworm with functional implications in pathogen recognition

C-type lectins can act as pattern recognition receptors (PRRs) in innate immunity. Previously, we identified two C-type lectins from silkworm (Bombyx mori), BmLBP and BmMBP, as PRRs. In the present study, we identified three homologs of these lectins by searching the silkworm genome database. These novel B. mori low-expression lectins were designated BmLEL-1, BmLEL-2, and BmLEL-3. Although Western-blot analysis failed to detect BmLEL-1, -2, or -3 in plasma, affinity precipitation of larval plasma with various microorganisms revealed that BmLEL-1 and -2 bind to rough and smooth strains of Gram-negative bacteria, respectively. BmLEL-1, -2, and -3 were found to be expressed in testis and ovary, where BmLEL-2 expression was up-regulated after bacteria infection. These results indicate that the novel C-type lectins might play a role in the innate immunity in these tissues as PRRs. Here, we discuss the roles and members of the C-type lectins as primary PRRs in B. mori cellular immunity.     

Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta

Neuroglian, a member of the L1 family of cell adhesion molecules (L1-CAMs), is expressed on surfaces of granular cells and a subset of large plasmatocytes of Manduca sexta that act as foci for hemocyte aggregation during the innate immune response. Neuroglian expressed on surfaces of transfected Sf9 cells induced their homophilic aggregation, with the aggregation being abolished in the presence of recombinant immunoglobulin (Ig) domains of neuroglian. Neuroglian and its Ig domains also can interact with hemocyte-specific integrin (HS integrin) as demonstrated with an enzyme-linked immunoassay and a surface plasmon resonance (SPR) assay. Neuroglian double-stranded (ds) RNA not only depresses expression of neuroglian in hemocytes but also depresses the cell-mediated encapsulation response of these hemocytes to foreign surfaces. After injection of a monoclonal antibody (MAb 3B11) into M. sexta larvae that recognizes the Ig domains of neuroglian, the cell-mediated encapsulation response of hemocytes was likewise inhibited. The Ig domains of neuroglian are involved in both homophilic and heterophilic interactions, and subsets of these six different Ig domains may affect different functions of neuroglian.     

Myeloid C-type lectins in innate immunity

C-type lectins expressed on myeloid cells comprise a family of proteins that share a common structural motif, and some act as receptors in pathogen recognition. But just as the presence of leucine-rich repeats alone is not sufficient to define a Toll-like receptor, the characterization of C-type lectin receptors in innate immunity requires the identification of accompanying signaling motifs. Here we focus on the known signaling pathways of myeloid C-type lectins and on their possible functions as autonomous activating or inhibitory receptors involved in innate responses to pathogens or self.