Activation of lobster hemocytes for phagocytosis

Activation of lobster (Homarus americanus) hemocytes for phagocytosis of sheep erythrocytes (SRBC) was demonstrated in vitro by incubation with lipopolysaccharide and by prolonged adherence to glass coverslips. Morphological changes, which preceded phagocytic activation, were detected by phase microscopy and Nomarski interference microscopy. These included spreading, the formation of filopodia and pseudopodia, granular darkening and dispersion, and vacuolation. Hemolymph serum opsonin greatly enhanced the recognition and phagocytosis of SRBC by activated hemocytes. Increases of 15 to 20 times background levels were observed both in the proportion of hemocytes which were actively phagocytic, and the percent of rosette-forming hemocytes. This suggested that the enhanced phagocytosis was the result of both the recruitment of a quiescent precursor population during activation, and an increase in the availability of opsonin binding sites on hemocyte membranes.     

C-type Lectin Binds to β-Integrin to Promote Hemocytic Phagocytosis in an Invertebrate

Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β-integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β-integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins.     

Identification of agglutinin receptors on hemocytes of Helix pomatia

The attachment of opsonized foreign particles to phagocytic cells indicates the occurrence of opsonin receptors on the surfaces of the phagocytes. There is good evidence that naturally occurring hemagglutinins may serve as opsonins in invertebrates. To prove the occurrence of agglutinin receptors on the hemocytes of an invertebrate, the interaction of various agglutinins with Helix pomatia hemocytes was investigated. A positive agglutination reaction was obtained with Ricinus, Axinella, anti-H_eel, Ulex, concanavalin A, and Limulus agglutinins. The known specificities of these agglutinins and the influence of carbohydrases on the agglutinability of Helix cells have led to the conclusion that the carbohydrate components of the binding sites include galactose, fucose, mannose or glucose or both, and N-acetylneuraminic acid or polygalactose.     

Effects of zinc on programmed cell death of Musca domestica and Drosophila melanogaster blood cells

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn²⁺, an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn²⁺, and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn²⁺ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn²⁺ also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.     

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6 h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.     

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus,Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.     

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A PEPTIDOGLYCAN RECOGNITION PROTEIN FROM THE COTTON BOLLWORM,Helicoverpa armigera

Peptidoglycan recognition proteins (PGRPs) specifically bind to peptidoglycans, and play crucial roles as pattern recognition receptors (PRRs) in mediating innate immune responses. In this study, we identified and characterized a PGRP (HaPGRP‐D) from the cotton bollworm, Helicoverpa armigera. Sequence analysis indicated that HaPGRP‐D is an amidase‐type PGRP. Expression of HaPGRP‐D was upregulated in the hemocytes of H. armigera larvae after injecting Gram‐negative Escherichia coli, Gram‐positive Staphylococcus aureus, or chromatography beads. To test the biological activity of HaPGRP‐D, purified recombinant protein was prepared. Subsequent analysis showed that rHaPGRP‐D (i) could bind and agglutinate Gram‐negative E. coli and Gram‐positive S. aureus in a zinc‐dependent manner, (ii) functioned as an amidase to degrade peptidoglycans in the presence of Zn²⁺, (iii) strongly inhibited the growth of E. coli and S. aureus in the presence of Zn²⁺, (iv) could bind to the surface of hemocytes, (v) increased the phagocytosis of E. coli cells by hemocytes in vitro, and (vi) promoted hemocyte encapsulation on chromatography beads in vitro. These results suggest that HaPGRP‐D plays important roles as PRR, amidase, and opsonin in H. armigera humoral and cellular immune responses.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Histopathology of Aerococcus viridans var. homari infection (Gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.     

The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2 ^−/− AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2 ^−/− mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.     

Insect Cytokine Paralytic Peptide (PP) Induces Cellular and Humoral Immune Responses in the Silkworm Bombyx mori

In the blood (hemolymph) of the silkworm Bombyx mori, the insect cytokine paralytic peptide (PP) is converted from an inactive precursor to an active form in response to the cell wall components of microorganisms and contributes to silkworm resistance to infection. To investigate the molecular mechanism underlying the up-regulation of host resistance induced by PP, we performed an oligonucleotide microarray analysis on RNA of blood cells (hemocytes) and fat body tissues of silkworm larvae injected with active PP. Expression levels of a large number of immune-related genes increased rapidly within 3 h after injecting active PP, including phagocytosis-related genes such as tetraspanin E, actin A1, and ced-6 in hemocytes, and antimicrobial peptide genes cecropin A and moricin in the fat body. Active PP promoted in vitro and in vivo phagocytosis of Staphyloccocus aureus by the hemocytes. Moreover, active PP induced in vivo phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in the fat body. Pretreatment of silkworm larvae with ML3403, a pharmacologic p38 MAPK inhibitor, suppressed the PP-dependent induction of cecropin A and moricin genes in the fat body. Injection of active PP delayed the killing of silkworm larvae by S. aureus, whereas its effect was abolished by preinjection of the p38 MAPK inhibitor, suggesting that p38 MAPK activation is required for PP-dependent defensive responses. These findings suggest that PP acts on multiple tissues in silkworm larvae and acutely activates cellular and humoral immune responses, leading to host protection against infection.     

In vitro bacteridical capacity of Blaberus craniifer hemocytes

Blaberus craniifer hemocytes, maintained in short-term culture, are capable of phagocytosing and destroying Staphylococcus aureus, Staphylococcus albus, Streptococcus faecalis, Serratia marcescens, and Proteus mirabilis. The observed bactericidal activity of the hemocyte suspensions was entirely a function of the phagocytes; the medium, the hemolymph, and cell products elaborated during incubations were not bactericidal. No humoral opsonic factors were required for, or facilitated, bacterial phagocytosis in vitro. Washed hemocyte monolayers bathed by hemolymph-free medium were capable of phagocytosing bacteria. The addition of hemolymph concentrated by ultrafiltration did not increase the bactericidal capacity of the hemocytes. Bacteria opsonized with concentrated hemolymph were not killed more efficiently than were untreated bacteria.A partial blockage of bactericidal capacity was induced by prior exposure of the hemocytes to bacteria or to latex particles. The functional blockade was more complete with bacteria than with latex particles.Pseudomonas aeruginosa, Escherichia coli, Salmonella typhosa, and Diplococcus pneumoniae were phagocytosed but not killed by the hemocytes. This lack of bactericidal activity suggests that roaches may encounter difficulty in eliminating these organisms from the hemocoel. However, deficient bactericidal capacity probably does not entirely correlate with pathogenicity since the known insect pathogens, Staphylococcus albus, Serratia marcescens, and Proteus mirabilis, are killed by the hemocytes. Pathogenicity seems to depend on a complex of factors including bacterial strain, dose received, and intracellular survival of ingested bacteria. A possible connection between the lack of hemocytic bactericidal capacity and the role of roaches as potential disease vectors warrants further investigation.     

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.     

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.     

A cell membrane-associated lectin of the oyster hemocyte

The presence of a lectin in association with hemocytes of the American oyster, Crassostrea virginica, has been demonstrated by utilizing a microhemagglutination assay. The plasma membrane association of this lectin is shown by its copurification with the plasma membrane fraction of disrupted hemocytes, using sucrose density gradient centrifugation, and also by the binding of ¹²⁵I-labeled glycoproteins to intact hemocytes at 4°C. Based upon agglutinating spcificity for a range of vertebrate erythrocytes, both untreated and enzyme-treated, along with hemagglutination-inhibition assays and crossed-absorption tests, it is apparent that there are also two serum (soluble) lectins, each having a distinct serological agglutination specificity, and that the hemocyte membrane-associated lectin has a specificity that is identical with one of these two serum lectins. It is proposed that the hemocyte membrane-associated lectin may be a true integral membrane protein, and therefore may function as a membrane receptor in nonself recognition by molluscan hemocytes.     

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum‐sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI‐independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester‐containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4‐mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild‐type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.