Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5–10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.

Nucleic acids, DNA and RNA, are universal in all living organisms and are polyanionic macromolecules that carry the whole genetic information of every cell. They are usually intracellular, but upon wounding or injury, nucleic acids are released from damaged tissue to the extracellular environment where they exert unexpected functions. In vertebrates, host- or pathogen-derived nucleic acids have been recognized as immunostimulatory factors because their extracellular presence induces IFN production by fibroblasts or immune cells (1, 2, 3). TLR-dependent (e.g., TLR3, TLR7, and TLR9) and TLR-independent (e.g., RIG-I/MDA5 and DNA-dependent activator of IFN-regulatory factors) signaling pathways have been described that are activated by nucleic acids from both pathogens and hosts (4, 5, 6, 7). In addition, human granular immune cells (neutrophils) were discovered to generate and to weave tangled webs of extracellular fibers composed of nucleic acids and proteins with antimicrobial capacities when stimulated by cytokines or bacterial immune elicitors like LPS (8). Because these webs are capable of entrapping microbes, they have been named neutrophil extracellular traps (NETs)2 (9, 10). The importance of this defense mechanism has, in parallel, been highlighted by the observation that bacterial pathogens expressing nucleic acid hydrolyzing enzymes are capable of escaping entrapment by these NETs during infection (11, 12, 13, 14).

We have recently demonstrated that extracellular nucleic acids derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage induce blood clotting in mammals (15). They may provide physiologically relevant templates for the factors XII/XI-induced contact activation/amplification during human hemostasis (16). Invertebrates lack acquired immunity, which evolved during vertebrate evolution, and rely on innate immunity to control pathogens. Therefore, we investigated potential functions of extracellular nucleic acids in innate immunity and hemolymph coagulation in a particularly suited insects model, the greater wax moth Galleria mellonella. G. mellonella caterpillars have widely been used as convenient and reliable model hosts for many insect and human pathogens (17, 18, 19, 20, 21, 22, 23, 24, 25). Among the advantages provided by the Galleria model, it is of particular importance to note that the caterpillars can be reared at mammalian physiological temperatures (around 37°C) to which human pathogens are adapted and which are essential for synthesis of many microbial virulence/pathogenicity factors. Furthermore, Galleria represents a classical model for the investigation on insect hemolymph clotting (26, 27, 28, 29), and it has recently been used to elucidate the mechanisms mediating sensing of infection by danger signals (30, 31, 32, 33), similar to signals proposed in the danger model of mammalian immunity (34).

The insect innate immune system recognizes microbe and damage associated pattern molecules by germline encoded receptors (e.g., Toll receptors and peptidoglycan recognition proteins) which engage potent defense reactions such as hemolymph coagulation, cellular phagocytosis, nodulation, encapsulation, and phenoloxidase activation leading to melanization (35). These reactions are often divided into cellular and humoral immune responses, although it is somewhat arbitrary, as many humoral factors affect hemocyte function and hemocytes are an important source of many humoral molecules. In Lepidoptera, granulocytes and plasmatocytes are the hemocyte types responsible for phagocytosis of microbes and become adherent upon stimulation (35, 36). The other hemocytes are nonadhesive spherule cells, oenocytoids, and prohemocytes (35, 36). Spherule cells have been suggested to transport cuticular components, while oenocytoids are fragile cells containg cytoplasmic phenoloxidase precursors that likely play a role in melanization of hemolymph. Prohemocytes are hypothesized to be stem cells that can differentiate into one or more of the aforementioned hemocyte types (35, 36).

In insects, killing of invading pathogens is achieved similar to mammals by enzymes (e.g., lysozymes), by reactive oxygen species, and by antimicrobial peptides (e.g., defensins) (35). These defense reactions rely on both the cellular and humoral immune responses. In this study, we report that survival of G. mellonella larvae infected with the entomopathogen Photorhabdus luminescens can be prolonged when host-derived extracellular nucleic acids are simultaneously injected in their hemocoels. This protective role was putatively mediated by the ability of host-derived nucleic acids to synergistically enhance both induced expression of antimicrobial peptides and activation of immune cells. Furthermore, we discovered that addition of extracellular RNA or DNA to hemolymph samples resulted in the formation of net-like coagulation fibers that efficiently entrap bacteria. Consequently, we addressed the question whether RNA is actively released by hemocytes upon immune stimulation, and from which could hemocyte type. Proteomic analyses revealed that corresponding RNA-binding proteins, particularly apolipoproteins, are potentially involved in nucleic acid-mediated defense reactions.

Galleria mellonella larvae were reared on an artificial diet (22% maize meal, 22% wheat germ, 11% dry yeast, 17.5% bee wax, 11% honey, and 11% glycerin) at 32°C in darkness. Photorhabdus luminescens strain DSM 12205 was purchased from DSMZ. Last instar larvae, each weighing between 250 and 350 mg, were used for injection experiments. Ten microliters of sample volume per larva were injected dorsolaterally into the hemocoel using 1-ml disposable syringes and 0.4 × 20 mm needles mounted on a microapplicator. Viable or heat-inactivated bacteria were washed three times in sterile PBS (20 mM Na3PO4 buffer, 100 mM NaCl (pH 7.0)) and subsequently mixed with DNA or RNA solutions immediately prior application.

RNA was extracted from whole larvae using the TriReagent isolation reagent (Molecular Research Centre) and Qiagen RNeasy kit (Qiagen) according to the instructions of the manufacturers. DNA was extracted from whole larvae using Qiagen DNeasy kit. Integrity of RNA and DNA was confirmed on ethidium bromide agarose gels, and quantities were determined spectrophotometrically (37). Hydrolysis of DNA (1 mg/ml) was performed with 1 μg/ml DNase I (Qiagen) 16 h at 37°C and of RNA (1 mg/ml) with 1 μg/ml RNase A (Qiagen) for 4 h at 60°C. Hydrolysis efficiencies were confirmed by ethidium bromide agarose gels (37).

Humoral antimicrobial activity was measured by an inhibition zone assay using a LPS-defective, streptomycin- and ampicillin-resistant mutant of E. coli K12 strain D31 (38, 39). Using the inhibition zone assay with Escherichia coli bacteria and gentamicin as an external standard, induction levels of antimicrobial peptides within the hemolymph 24 h post treatment were quantified which is a degree of the systemic immune responses in insects (32). Cellular immune stimulation in vivo was determined by counting circulating hemocytes in the hemolymph 4 h upon treatment of larvae because immune stimulation correlates with the switch from nonadherent, resting hemocytes to activated, adherent cells (40).

Cell-free hemolymph samples were isolated by bleeding injected larvae 2 h post injection, or untreated larvae, into plastic tubes with traces of phenylthiourea to prevent phenoloxidase activation followed by a centrifugation step of the hemolymph at 2,000 × g for 5 min. The supernatant (cell-free hemolymph) was precipitated by the addition of 3 volumes of 100% acetone and 0.4 volumes of 100% trichloroacetic acid followed by incubation at −20°C for 1 h. After centrifugation at 20,000 × g for 10 min, the pellet was washed three times with 100% acetone and resolved under agitation in 8 M urea at 22°C for 16 h. Protein concentrations were determined using the Micro BC assay kit (Uptima). Two-dimensional gel electrophoresis, in-gel digestion, and protein identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry were performed as described (32).

Cell-free hemolymph samples from LPS-challenged larvae were isolated by bleeding larvae 24 h post injection with 100 μg LPS (Sigma-Aldrich) similarly as described (32). Obtained hemolymph proteins were separated by Tris-Tricine-SDS-PAGE and blotted on to a polyvinylidene difluoride (PVDF) membrane according to the manufacturer’s instructions (Amersham Biosciences). Apolipoprotein III (ApoLp-III) was partially purified as described (41) prior to SDS-PAGE and blotting on the PVDF membrane. After precipitation of most other hemolymph proteins by heat treatment at 90°C for 30 min, the supernatant was obtained by centrifugation at 20,000 × g for 30 min. The supernatant is mainly composed of heat-stable arylphorin protein (∼80 kDa) and apolipoprotein III (41). Membranes were incubated in buffer A containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, and 1× Denhardt’s solution (0.02% weight-to-volume ratio (w/v) Ficoll, 0.02% w/v,polyvinylpyrrolidone, and 0.02% w/v BSA) at 4°C overnight. Subsequently, the membranes were incubated 1 h at 25°C in 30 ml buffer A containing 3 μg biotin-labeled total RNA. Labeling of total RNA was conducted using EZ-Link Psoralen-PEO3-Biotin (Pierce) according to the manufacturer’s instructions. After washing overnight with buffer A, the membranes were subjected to chemiluminescent detection using x-ray films (Amersham Biosciences) and the SuperSignal West Pico Complete Biotinylated Protein Detection Kit (Pierce) according to the instructions of the manufacturers.

Larvae were punctured with a sterile needle, and obtained hemolymph samples (∼10 μl) were dropped directly on test solutions (5 μl) on the microscope slides. Coagulation was monitored with an Axioplan 2 microscope (Zeiss). DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) (Sigma-Aldrich) and SYTOX Green were used for staining of nucleic acids according to the instructions of the manufacturers. FITC-labeled bacteria were prepared by coupling FITC to amine groups of bacteria using fluoresceinisothiocyanate similar as proposed by the manufacturers’ instructions (KMF Laborchemie Handels-GmbH).

To analyze potential functions of host-derived nucleic acids in Galleria, we tested their immunostimulatory activities. Using the inhibition zone assay with Escherichia coli bacteria and gentamicin as an external standard, induction levels of antimicrobial peptides within the hemolymph 24 h post treatment were quantified, which correlate to the activation level of the systemic immune response in insects (32). Injection of purified RNA (mainly composed of rRNA and mRNA with an average chain length of approximate 1000–4000 nt) alone, up to 20 μg per animal, did not result in significant systemic immune responses (gentamicin aquivalents 2.5 ± 0.5 U/ml) when compared with PBS injected animals (gentamicin aquivalents 2.4 ± 0.4 U/ml) (Fig. 1,A). However, a synergistically increased immune response was observed in larvae that had been injected with 10 μg RNA along with heat-killed P. luminescens bacteria (gentamicin aquivalents 13 ± 0.8 U/ml) when compared with larvae that had been injected with PBS along with heat-killed P. luminescens bacteria injected animals (gentamicin aquivalents 7.9 ± 0.9 U/ml) (Fig. 1 A). The difference was statistically significant (p < 0.001) as calculated with a Student’s t test. Furthermore, this synergistic effect was abolished when RNase was coinjected along with RNA and heat-killed bacteria (gentamicin aquivalents 8.5 ± 0.8 U/ml).

FIGURE 1.

Extracellular presence of RNA synergistically induces humoral and cellular immune responses against bacteria. A, Induced systemic expression levels of antimicrobial peptides in the hemolymph is shown as gentamicin equivalents (U/ml were calculated using a calibration curve with gentamicin). RNA injection did not result in significant systemic immune responses leading to increased antimicrobial peptide levels in larval hemolymph (gentamicin aquivalents 2.5 ± 0.5 U/ml) when compared with PBS injection (gentamicin aquivalents 2.4 ± 0.4 U/ml). Larvae that had been injected with 10 μg RNA along with heat-killed P. luminescens bacteria (gentamicin aquivalents 13 ± 0.8 U/ml) have a synergistically increased immune response when compared with PBS plus heat-killed P. luminescens bacteria injected animals (gentamicin equivalents 7.9 ± 0.9 U/ml). This effect was abolished when RNase was coinjected along with RNA and heat-killed bacteria (gentamicin aquivalents 8.5 ± 0.8 U/ml). B, In vivo hemocyte stimulation was quantified by the depletion rate of circulating nonactivated hemocytes. A synergistically increased cellular immune stimulation was observed when 10 μg RNA were coinjected with heat-killed P. luminescens bacteria (0.78 ± 0.18 × 103 hemocytes/μl) when compared with injected bacteria alone (1.26 ± 0.34 × 103 hemocytes/μl). This activation of the cellular immunity was abolished when the RNA had been hydrolyzed by RNase A prior use (1.1 ± 0.28 × 103 hemocytes/μl). RNA injection alone resulted in no significant cellular immune activation (3.9 ± 0.7 × 103 hemocytes/μl) when compared with PBS injection (4 ± 0.57 × 103 hemocytes/μl). Results represent mean values of at least three independent determinations ± SD. Statistically significant differences were determined using Student‘s t test and are indicated (∗, p < 0.05; ∗∗∗, p < 0.005).

FIGURE 1.

Extracellular presence of RNA synergistically induces humoral and cellular immune responses against bacteria. A, Induced systemic expression levels of antimicrobial peptides in the hemolymph is shown as gentamicin equivalents (U/ml were calculated using a calibration curve with gentamicin). RNA injection did not result in significant systemic immune responses leading to increased antimicrobial peptide levels in larval hemolymph (gentamicin aquivalents 2.5 ± 0.5 U/ml) when compared with PBS injection (gentamicin aquivalents 2.4 ± 0.4 U/ml). Larvae that had been injected with 10 μg RNA along with heat-killed P. luminescens bacteria (gentamicin aquivalents 13 ± 0.8 U/ml) have a synergistically increased immune response when compared with PBS plus heat-killed P. luminescens bacteria injected animals (gentamicin equivalents 7.9 ± 0.9 U/ml). This effect was abolished when RNase was coinjected along with RNA and heat-killed bacteria (gentamicin aquivalents 8.5 ± 0.8 U/ml). B, In vivo hemocyte stimulation was quantified by the depletion rate of circulating nonactivated hemocytes. A synergistically increased cellular immune stimulation was observed when 10 μg RNA were coinjected with heat-killed P. luminescens bacteria (0.78 ± 0.18 × 103 hemocytes/μl) when compared with injected bacteria alone (1.26 ± 0.34 × 103 hemocytes/μl). This activation of the cellular immunity was abolished when the RNA had been hydrolyzed by RNase A prior use (1.1 ± 0.28 × 103 hemocytes/μl). RNA injection alone resulted in no significant cellular immune activation (3.9 ± 0.7 × 103 hemocytes/μl) when compared with PBS injection (4 ± 0.57 × 103 hemocytes/μl). Results represent mean values of at least three independent determinations ± SD. Statistically significant differences were determined using Student‘s t test and are indicated (∗, p < 0.05; ∗∗∗, p < 0.005).

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We obtained similar synergistic effects when we examined hemocyte stimulation. To quantify cellular immune stimulation, we counted circulating hemocytes in the hemolymph of animals 4 h after injection with different solutions. Depletion of circulating nonactivated hemocytes indicate cellular stimulation in vivo because immune activated hemocytes become highly adhesive and attach to each other forming multicellular aggregates and to internal organs of the larvae (40). When 10 μg RNA were coinjected with heat-killed P. luminescens bacteria, an increased cellular innate immune response was determined (hemocyte count of 0.78 ± 0.18 × 103 cells/μl) when compared with animals injected with bacteria along with PBS (hemocyte count of 1.26 ± 0.34 × 103 cells/μl) (Fig. 1,B). The difference was statistically significant (p = 0.02) as determined with a Student’s t test. This effect was abolished when the RNA had been hydrolyzed by RNase treatment prior to use (hemocyte count of 1.1 ± 0.28 × 103 cells/μl). RNA injection alone resulted in no significant cellular immune activation (hemocyte count of 3.9 ± 0.7 × 103 cells/μl) when compared with PBS injection (hemocyte count of 4 ± 0.57 × 103 cells/μl) (Fig. 1 B).

Moreover, high amounts of RNA (more than 50 μg per larvae) resulted in activation of the prophenoloxidase cascade in vivo, a serine-proteinase cascade, leading to melanisation of hemolymph in the larvae (data not shown). However, the biological significance of this prophenoloxidase cascade activation at high concentrations of nucleic acids is not clear and will be investigated in future studies. In general, similar immunostimulatory activities were determined when DNA was used instead of RNA (data not shown). The DNA mediated effects were also abolished when DNA had been degraded by DNA hydrolyzing enzyme prior to injection as similarly shown above for RNA.

In a next step, we investigated the procoagulant potential of nucleic acids. Larvae were pierced with a sterile needle, and hemolymph samples (∼10–15 μl) collected from the wound were directly applied on microscope slides containing 5 μl of test solutions with LPS, water, or nucleic acids. Within minutes, the formation of net-like structures were induced by RNA and DNA, respectively, in the hemolymph (Fig. 2, C and D). This nucleic acid-dependent process appeared to be similar to the LPS-induced clotting reactions that we determined in our analysis (Fig. 2 B). In contrast, coagulation induction was reduced when nucleic acids had been hydrolyzed prior to analysis because less coagulation strands could be observed under the microscope within 5–10 min (data not shown).

FIGURE 2.

The presence of nucleic acids induces the formation of net-like fibrillar coagulation strands in the insect hemolymph. A, Larvae were pierced with a sterile needle, and hemolymph samples collected from the wound were directly applied on microscope slides containing 5 μl water (A) or solutions of 1 mg/ml LPS (B), RNA (C), or DNA (D). Within minutes the formation of net-like structures were detected (indicated by arrows) that bind to surrounding hemocytes. Differential interference contrast (Nomarski) image, × 100. Scale bars, 50 μm.

FIGURE 2.

The presence of nucleic acids induces the formation of net-like fibrillar coagulation strands in the insect hemolymph. A, Larvae were pierced with a sterile needle, and hemolymph samples collected from the wound were directly applied on microscope slides containing 5 μl water (A) or solutions of 1 mg/ml LPS (B), RNA (C), or DNA (D). Within minutes the formation of net-like structures were detected (indicated by arrows) that bind to surrounding hemocytes. Differential interference contrast (Nomarski) image, × 100. Scale bars, 50 μm.

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To identify host proteins that directly interact with and thereby mediate the enhancing effects of extracellular nucleic acids on humoral and cellular immune responses a combination of proteomic approaches was used. First, Galleria hemolymph samples obtained from LPS-challenged larvae were analyzed by Northwestern blot analysis. Five protein bands within the hemolymph were detected that interact with labeled RNA (Fig. 3, lane 2). The RNA binding protein with ∼17 kDa may correspond to apolipoprotein-III (ApoLp-III), whose role in β 1,3-glucan pattern recognition and cellular encapsulation in G. mellonella larvae has recently been established by the group of Norman Ratcliffe (42). To confirm its identity ApoLp-III was partially purified by precipitation of most other hemolymph proteins by heat treatment. The supernatant still contained binding activity of ApoLp-III for RNA as demonstrated by Northwestern blot analysis (data not shown). In addition, it has recently been demonstrated that ApoLp-II forms partially SDS-stable complexes with ApoLp-III in the Galleria hemolymph with a molecular mass of 80–90 kDa (43). These stable complexes may correspond to the detected protein band at ∼85 kDa in the Northwestern blot analysis (Fig. 3, lane 2). The identities of the proteins with an estimated molecular mass of 70 and 50 kDa, respectively, remained unclear.

FIGURE 3.

Interaction of Galleria hemolymph proteins with labeled RNA. Galleria hemolymph proteins obtained from LPS-challenged animals were separated by Tris-tricine-SDS-PAGE and blotted on to a PVDF membrane. Proteins were stained with Coomassie Blue, photographed (lane 1), completely destained with 70% (v/v) ethanol, and equilibrated in binding buffer. The membrane was incubated with biotin-labeled total RNA. Labeled RNA bound to the interacting proteins on the membrane was detected by chemoluminescent reaction and x-ray films, resulting in dark bands (lane 2). The protein interacting with RNA at ∼17 kDa corresponds to apolipoprotein-III (ApoLp-III). The potential SDS-stable complex of ApoLp-II and ApoLp-III (∼85 kDa) is indicated by an asterisk. The bands corresponding to 50 and 70 kDa proteins are not known. Molecular mass standards are shown in kDa.

FIGURE 3.

Interaction of Galleria hemolymph proteins with labeled RNA. Galleria hemolymph proteins obtained from LPS-challenged animals were separated by Tris-tricine-SDS-PAGE and blotted on to a PVDF membrane. Proteins were stained with Coomassie Blue, photographed (lane 1), completely destained with 70% (v/v) ethanol, and equilibrated in binding buffer. The membrane was incubated with biotin-labeled total RNA. Labeled RNA bound to the interacting proteins on the membrane was detected by chemoluminescent reaction and x-ray films, resulting in dark bands (lane 2). The protein interacting with RNA at ∼17 kDa corresponds to apolipoprotein-III (ApoLp-III). The potential SDS-stable complex of ApoLp-II and ApoLp-III (∼85 kDa) is indicated by an asterisk. The bands corresponding to 50 and 70 kDa proteins are not known. Molecular mass standards are shown in kDa.

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Because LPS has been shown to mediate the formation of ApoLp-I/II/III-containing lipoprotein aggregates in Galleria (44), nucleic acids were tested for similar activities in the hemolymph. In accordance with published information (44), 20 μg/ml LPS induced the formation of detergent-stable aggregates in cell-free diluted hemolymph which correlated well with the decrease of ApoLp-I and ApoLp-II protein bands (Fig. 4,A, lanes 1 and 2). Comparable aggregate formation was found in the presence of 20 μg/ml RNA or DNA (Fig. 4,A, lanes 3 and 5). As expected, presence of RNase or DNase, respectively, prevented enhanced aggregate formation (Fig. 4 A, lane 4 and 6). Furthermore, the reducing agent 2-ME inhibited both LPS- and nucleic acid-mediated lipoprotein aggregate formation indicating that processes that are essential for defense reactions are activated in a similar way by an endogenous danger signal (nucleic acids) and an exogenous immune elicitor (LPS).

FIGURE 4.

Proteomic analysis of G. mellonella hemolymph proteins in the presence of nucleic acids. A, Incubation of 1 to 100 diluted Galleria cell-free hemolymph in 120 mM NaCl and 20 mM Na-phosphate buffer (pH 7.0) for 16 h at 25°C results in the formation of a small amount of SDS-stable aggregates (lane 1) which are strongly enhanced in the presence of 20 μg/ml LPS (lane 2) similar as also reported by others (31 ). The induced aggregate formation correlates well with decrease of ApoLp-I (∼ 250 kDa) and ApoLp-II (∼ 85 kDa) bands (indicated by arrows). Comparable aggregate formation is detected in the presence of 20 μg/ml RNA (lane 3) and DNA (lane 5), respectively. The DNA/RNA-induced aggregate formation is abolished in the presence of enzymes that hydrolyze nucleic acids (lanes 4 and 6). The presence of reducing agent (2 mM 2-ME) resulted in complete inhibition of aggregate formation by nucleic acids and LPS (data not shown). B, Hemolymph protein samples (1 mg) from 30 untreated and 30 RNA injected larvae were loaded on 24-cm pH 3–11 NL-IEF strips followed by Tris-tricine-SDS-PAGE on a 15% gel, respectively. Four out of ∼500 abundant hemolymph proteins with increased and one with reduced abundance after 2 h RNA-injection were identified. Spot 1 may correspond to ApoLp-II because ApoLp-II is an abundant hemolymph protein with an estimated molecular mass of 85 kDa. Molecular mass standards are indicated in kDa.

FIGURE 4.

Proteomic analysis of G. mellonella hemolymph proteins in the presence of nucleic acids. A, Incubation of 1 to 100 diluted Galleria cell-free hemolymph in 120 mM NaCl and 20 mM Na-phosphate buffer (pH 7.0) for 16 h at 25°C results in the formation of a small amount of SDS-stable aggregates (lane 1) which are strongly enhanced in the presence of 20 μg/ml LPS (lane 2) similar as also reported by others (31 ). The induced aggregate formation correlates well with decrease of ApoLp-I (∼ 250 kDa) and ApoLp-II (∼ 85 kDa) bands (indicated by arrows). Comparable aggregate formation is detected in the presence of 20 μg/ml RNA (lane 3) and DNA (lane 5), respectively. The DNA/RNA-induced aggregate formation is abolished in the presence of enzymes that hydrolyze nucleic acids (lanes 4 and 6). The presence of reducing agent (2 mM 2-ME) resulted in complete inhibition of aggregate formation by nucleic acids and LPS (data not shown). B, Hemolymph protein samples (1 mg) from 30 untreated and 30 RNA injected larvae were loaded on 24-cm pH 3–11 NL-IEF strips followed by Tris-tricine-SDS-PAGE on a 15% gel, respectively. Four out of ∼500 abundant hemolymph proteins with increased and one with reduced abundance after 2 h RNA-injection were identified. Spot 1 may correspond to ApoLp-II because ApoLp-II is an abundant hemolymph protein with an estimated molecular mass of 85 kDa. Molecular mass standards are indicated in kDa.

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In a subsequent step, RNA was injected into larvae to analyze changes of hemolymph proteins in vivo. As compared with control animals, the spectrum of ∼500 most abundant hemolymph proteins was analyzed by two-dimensional gel electrophorsis. In agreement with data obtained in vitro, a dominant protein spot at ∼80 kDa disappeared 2 h post RNA injection that may most probably correspond to ApoLp-II and, additionally, four other protein spots were found to be induced in the hemolymph (Fig. 4 B). Unfortunately, matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis resulted in no positive identifications because the corresponding sequences from these Galleria proteins were probably not yet in public databases. In addition, the two-dimensional protein map from G. mellonella larvae 24 h post RNA injection showed no significant differences to hemolymph proteins from untreated animals (data not shown) suggesting no induced or repressed expression of abundant hemolymph proteins 24 h upon RNA injection in Galleria.

Using SYTOX Green, a selective dye that visualizes the extracellular presence of nucleic acids, we searched for intrinsic nucleic acids during insect clotting. We performed an ex vivo analysis without any permeabilization or fixation steps by directly applying hemolymph samples (10–15 μl) collected from the wound site of larvae on microscope slides containing 5 μl of test solutions with appropriate stains or FITC-labeled bacteria. We found that Galleria oenocytoids which constitute ∼5–10% of total circulating hemocytes represent a source of endogenously derived extracellular nucleic acids in the hemolymph. Oenocytoids rupture after 10 to 60 s upon immune stimulation by bleeding on microscope slides and are the primary source for extracellular nucleic acids as demonstrated by SYTOX staining (Fig. 5, A and B). Within 1–3 min, the granulocytes became degranulated most probably triggered by oenocytoid-derived factors including extracellular nucleic acids. Degranulation of granulocytes results in efficient microbial entrapment at their surface and at fibrillar structures appearing between these cells (Fig. 5, C and D). Examination of the hemolymph by the hanging drop approach (26) led to the observation that within 3–5 min coagulation strands including hemocytes and bacteria were formed (Fig. 5, E and F). Additionally, within 5–15 min multicell aggregations with increased activation of the serine proteinase cascade leading to melanisation were detected (Fig. 5,G). DAPI staining under used conditions resulted in strong fluorescence of “naked” nuclei of oenocytoids and of faint staining of nuclei of other hemocytes which are covered by a surrounding cell membrane (Fig. 5,H). To identify potential NETs formed by insect cells, we incubated hemolymph samples (10 μl) mixed with Schneider’s insect medium (200 μl) (BioWhittaker) containing FITC-labeled bacteria and DAPI stain on microscope slides in a humidity chamber for 2–5 h. Hemocytes coagulated with hemolymph proteins and bacteria but no cells were found producing NETs as known from human neutrophils (Fig. 5, I and J).

FIGURE 5.

Detection of intrinsic extracellular nucleic acids derived from oenocytoids during insect hemolymph clotting. A, Microscopic examination of Galleria hemolymph samples containing live hemocytes resulted in the observation that oenocytoids (Oc) rupture within 10–60 s upon bleeding. B, The rupture of oenocytoids results in visible fibrillar strands that contain nucleic acids because they are positively stained by SYTOX Green nucleic acid stain (indicated by an arrow). The “naked” nucleus is intensively stained. The granulocyte (Gc) is unstained because the cell is intact. C and D, Within 1–3 min the granulocytes degranulate substances that entrap FITC-labeled bacteria and results in fibrillar net-like coagulation structures. E and F, By the hanging drop method (26 ) we found 3–5 min after bleeding that coagulation strands developed including hemocytes and entrapped FITC-labeled bacteria. G, Within 5–15 min we observed multicell aggregates with enhanced activation of the phenoloxidase cascade resulting in melanisation (brownish color). H, In these aggregates we detected intense DAPI staining of free cell nuclei of ruptured oenocytoids and faint nuclei staining of intact cells. I, To identify NETs from insect cells we incubated hemolymph samples for 2–5 h in Schneider’s insect medium in the presence of FITC-labeled bacteria and DAPI stain. Hemocytes coagulated with hemolymph proteins and bacteria but no cells were found producing NETs as known from human neutrophils. J, Overlay of coagulon including DAPI stained nuclei and FITC-labeled bacteria is shown. Aggregated and fragmented nuclei of granulocytes (Gc) and plasmatocytes (Pc) (shown magnified in insets) indicate apoptotic processes after 3–4 h stimulation whereas “naked” nuclei of oenocytoids (Oc) are highly condensed during incubation period. Note that many bacteria are entrapped by granulocytes and only few are bound to plasmatocytes. Differential interference contrast (Nomarski) and fluorescence imaging; A–D and insets in J, ×1.575; E–J, ×630. Scale bars, 10 μm.

FIGURE 5.

Detection of intrinsic extracellular nucleic acids derived from oenocytoids during insect hemolymph clotting. A, Microscopic examination of Galleria hemolymph samples containing live hemocytes resulted in the observation that oenocytoids (Oc) rupture within 10–60 s upon bleeding. B, The rupture of oenocytoids results in visible fibrillar strands that contain nucleic acids because they are positively stained by SYTOX Green nucleic acid stain (indicated by an arrow). The “naked” nucleus is intensively stained. The granulocyte (Gc) is unstained because the cell is intact. C and D, Within 1–3 min the granulocytes degranulate substances that entrap FITC-labeled bacteria and results in fibrillar net-like coagulation structures. E and F, By the hanging drop method (26 ) we found 3–5 min after bleeding that coagulation strands developed including hemocytes and entrapped FITC-labeled bacteria. G, Within 5–15 min we observed multicell aggregates with enhanced activation of the phenoloxidase cascade resulting in melanisation (brownish color). H, In these aggregates we detected intense DAPI staining of free cell nuclei of ruptured oenocytoids and faint nuclei staining of intact cells. I, To identify NETs from insect cells we incubated hemolymph samples for 2–5 h in Schneider’s insect medium in the presence of FITC-labeled bacteria and DAPI stain. Hemocytes coagulated with hemolymph proteins and bacteria but no cells were found producing NETs as known from human neutrophils. J, Overlay of coagulon including DAPI stained nuclei and FITC-labeled bacteria is shown. Aggregated and fragmented nuclei of granulocytes (Gc) and plasmatocytes (Pc) (shown magnified in insets) indicate apoptotic processes after 3–4 h stimulation whereas “naked” nuclei of oenocytoids (Oc) are highly condensed during incubation period. Note that many bacteria are entrapped by granulocytes and only few are bound to plasmatocytes. Differential interference contrast (Nomarski) and fluorescence imaging; A–D and insets in J, ×1.575; E–J, ×630. Scale bars, 10 μm.

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To elucidate in vivo roles of extracellular RNA/DNA in insect immune defense, the influence of host-derived nucleic acids during infection of Galleria larvae with Photorhabdus bacteria was analyzed. Survival times of larvae receiving 10 μg RNA in combination with 103 CFU of Photorhabdus cells were significantly longer (50% mortality occurred at ∼37 h) than those of controls that were injected with bacteria alone (50% mortality at ∼21 h) or with bacteria plus RNA that had been hydrolyzed prior injection (50% mortality at ∼21 h) (Fig. 6,A). A similar protective effect was observed when DNA was coinjected along with bacteria resulting in a 50% mortality of larvae at ∼36 h (data not shown). However, when a 100-fold higher inoculum of bacteria was used (105 CFU of Photorhabdus cells per larvae), the prolonged survival rate in the presence of 10 μg RNA was only marginally reduced with a 50% mortality at ∼18 h vs 50% mortality at ∼16 h without RNA (Fig. 6 B). A further control group injected with heat-killed bacteria resulted in 100% survival, indicating that injection injury itself did not cause mortality.

FIGURE 6.

Survival curve of G. mellonella larvae infected with P. luminescens. A, All Galleria larvae (n = 10) that have been injected with P. luminescens (103 CFU/larvae) died after 24 h incubation at 32°C (○). Injection of 10 μg RNA along with P. luminescens significantly prolonged survival of infected Galleria larvae up to 35 to 42 h (•). B, Prolonged survival of larvae following their treatment was only marginal when a 100-fold higher inoculum of P. luminescens (105 CFU/larvae) was injected along with RNA. There was no killing of caterpillars that received heat-killed bacterial cells of the same strain. The experiment was repeated at least three times, with similar results.

FIGURE 6.

Survival curve of G. mellonella larvae infected with P. luminescens. A, All Galleria larvae (n = 10) that have been injected with P. luminescens (103 CFU/larvae) died after 24 h incubation at 32°C (○). Injection of 10 μg RNA along with P. luminescens significantly prolonged survival of infected Galleria larvae up to 35 to 42 h (•). B, Prolonged survival of larvae following their treatment was only marginal when a 100-fold higher inoculum of P. luminescens (105 CFU/larvae) was injected along with RNA. There was no killing of caterpillars that received heat-killed bacterial cells of the same strain. The experiment was repeated at least three times, with similar results.

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Our study identifies extracellular nucleic acids (naturally released by damaged tissues and by activated oenocytoids) as a novel danger signal in defense reactions of insects to protect them against infecting bacteria. The role of nucleic acids as alarm signals in antimicrobial defense is mediated by their capacity to synergistically induce humoral and cellular immune responses during infection and to induce net-like coagulation structures that entrap invading microbes. Results obtained by two-dimensional gel electrophoresis and Northwestern-blot analyses identified apolipoproteins as potential components involved in these processes.

The first defense response to wounding in insects is hemolymph coagulation. This clotting reaction shares functional similarities with vertebrate hemostasis: to seal wounds and to prevent life-threatening loss of blood (35, 45). In vertebrates, blood and lymph are confined to vessels, whereas in insects an open circulatory system provides access of hemolymph to other tissue cells. Yet, in both systems, the contribution of circulating cells is of great importance for efficient clot formation to occur (26, 46). Upon wounding, circulating hemocytes in insects immediately switch from a resting, nonadherent state to activated cells that are highly adhesive like activated platelets in mammals. However, unlike platelets, insect hemocytes contribute to microbial clearance by engulfment within multicellular aggregate formation, known as nodules with subsequent melanization. Although mechanisms that initiate these processes are hardly defined, we propose that extracellular nucleic acids serve as important, but as yet unrecognized, cofactors.

In this study, we discovered that oenocytoids release nucleic acids upon immune activation. Oenocytoids in Lepidoptera show similarities to the crystal cells in the fruit fly Drosophila melanogaster because both are large, regular in shape, contain phenoloxidases, and rupture upon immune activation (35, 36, 47). However, Drosophila has obviously no granulocytes and no apolipoprotein III gene which is present in many other insects (48), suggesting striking differences between different insect species. Because insects occupy a wide range of ecological niches, comparative studies using Galleria and other insects as models should enhance our understanding of blood cell-dependent innate immune responses in terms of conserved and derived molecular mechanisms.

In a previous study, we provided evidence that extracellular nucleic acids, in particular RNA, represent the long-sought natural “foreign surface” in the vertebrate system to induce blood clotting, particularly under pathological conditions (15). In analogy, we show in this study that the exposure of isolated hemolymph samples from G. mellonella larvae to extracellular RNA or DNA resulted in hemocyte activation and formation of net-like coagulation structures. Our results provide evidence that extracellular nucleic acids derived from ruptured oenocytoids or tissue damage trigger coagulation and other immune defense mechanisms in Galleria.

Insect coagulation nets share similarities in appearance and function (entrapment of microbes) with the indicated vertebrate NETs (27, 49, 50, 51, 52) but have more similarities to mammalian fibrin-platelet matrix. In contrast to human NETs, the extracellular nucleic acids in Galleria exhibit only weak bacterial entrapment capacity when compared with the binding capacity of the matrix that derives from granulocytes (Fig. 5, C and D). Furthermore, oenocytoids release nucleic acids within seconds upon stimulation whereas vertebrate NET formation is a slow process. In general, 180 min upon activation, NET components (including nucleic acids and cellular proteins) mix freely in the neutrophils and 15–60 min after that, NETs are released (9). Our results indicate that extracellular nucleic acids are at least as important as microbial derived molecular patterns for inducing hemolymph coagulation and melanization in vivo and provide a novel link between the vertebrate and insect system in terms of molecular principles that lead to engagement of the innate immunity.

Consistent with our results, a recent study demonstrates that mammalian LL37, an antimicrobial peptide released during skin injury, converts self-DNA into a “danger signal” that potently activates innate immune responses (53). On the other side, circulating nucleic acids in plasma and serum are prognostic markers for stroke, infarct, or cancer patients (54, 55), and it was recently shown that in DNase II-deficient mice, extracellular DNA escapes degradation and may cause chronic polyarthritis resembling human rheumatoid arthritis (56). These observations along with our results from insects indicate that extracellular nucleic acids exhibit evolutionarily conserved protective roles in the first line of defense which may turn to harmful effects when their degradation is dysregulated.

We found that apolipoproteins are potentially involved in nucleic acid-mediated hemocyte activation and coagulation in insects. Insect ApoLp-III is a multifunctional protein that is involved in lipid transport but also functions as pattern recognition receptor by binding to LPS, lipoteichoic acid, or to fungal β 1,3-glucan to induce cellular and humoral immune responses (42). Interestingly, mammalian apolipoproteins are analogously involved in LPS binding and other roles in innate immunity (57). Because insect ApoLp-III and ApoLp-I/II is homologous to human apolipoprotein E (58) and human apolipoprotein B (59), respectively, our results favor these molecules to be involved in the early defense mechanisms in vertebrates as well and provides a starting point for the investigation of interrelationships of innate immunity and physiological processes such as lipid storage and use.

The present findings are consistent with the hypothesis that extracellular nucleic acids promote host survival by improving defense mechanisms against pathogens at sites of tissue damage/infection in both insects and mammals (Fig. 7). Our data confirm that Galleria is a powerful model organism for analyzing host defense reactions in general and the role of extracellular nucleic acids in innate immunity in particular.

FIGURE 7.

Schematic overview of comparable roles of extracellular nucleic acids in insects and mammals.

FIGURE 7.

Schematic overview of comparable roles of extracellular nucleic acids in insects and mammals.

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We thank Meike Fischer for excellent technical assistance, Katja Altincicek for critical reading of the manuscript, and Monica Linder for MALDI-TOF-MS analysis.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

Abbreviations used in this paper: NET, neutrophil extracellular trap; w/v, weigh to volume ratio; PVDF, polyvinylidene difluoride membrane.

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