This information is current as
of June 15, 2017.
Host-Derived Extracellular Nucleic Acids
Enhance Innate Immune Responses, Induce
Coagulation, and Prolong Survival upon
Infection in Insects
Boran Altincicek, Sabine Stötzel, Malgorzata Wygrecka,
Klaus T. Preissner and Andreas Vilcinskas
J Immunol 2008; 181:2705-2712; ;
doi: 10.4049/jimmunol.181.4.2705
http://www.jimmunol.org/content/181/4/2705
Subscription
Permissions
Email Alerts
This article cites 58 articles, 17 of which you can access for free at:
http://www.jimmunol.org/content/181/4/2705.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
References
The Journal of Immunology
Host-Derived Extracellular Nucleic Acids Enhance Innate
Immune Responses, Induce Coagulation, and Prolong Survival
upon Infection in Insects
Boran Altincicek,* Sabine Stötzel,* Malgorzata Wygrecka,† Klaus T. Preissner,†
and Andreas Vilcinskas1*
N
ucleic 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 (1–3). TLRdependent (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 –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
*Institute of Phytopathology and Applied Zoology and †Institute of Biochemistry,
Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, Giessen, Germany
Received for publication April 25, 2008. Accepted for publication June 10, 2008.
(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 (11–14).
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 (17–25).
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 –29), and it has recently been used to elucidate the mechanisms mediating sensing of infection by danger
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.
1
Address correspondence and reprint requests to Dr. Andreas Vilcinskas, Institute
of Phytopathology and Applied Zoology, Justus-Liebig-University of Giessen,
Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany. E-mail address:
[email protected]
www.jimmunol.org
2
Abbreviations used in this paper: NET, neutrophil extracellular trap; w/v, weigh to
volume ratio; PVDF, polyvinylidene difluoride membrane.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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. The Journal of Immunology, 2008, 181: 2705–2712.
2706
EXTRACELLULAR NUCLEIC ACIDS IN INSECT INNATE IMMUNITY
Materials and Methods
Insect rearing and manipulation
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.
Preparation of RNA and DNA from G. mellonella larvae
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 ␮g/ml DNase I (Qiagen) 16 h at 37°C and of RNA (1
mg/ml) with 1 ␮g/ml RNase A (Qiagen) for 4 h at 60°C. Hydrolysis efficiencies were confirmed by ethidium bromide agarose gels (37).
Determination of humoral and cellular immune responses
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).
Two-dimensional gel electrophoresis of hemolymph proteins
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).
Northwestern blot analysis
Cell-free hemolymph samples from LPS-challenged larvae were isolated
by bleeding larvae 24 h post injection with 100 ␮g 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 ␮g 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.
Coagulation assay
Larvae were punctured with a sterile needle, and obtained hemolymph
samples (⬃10 ␮l) were dropped directly on test solutions (5 ␮l) 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).
Results
Extracellular nucleic acids enhance systemic and cellular innate
immune responses and hemolymph coagulation in insects
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
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
signals (30 –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.
The Journal of Immunology
2707
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 ␮g 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. 1A). However, a synergistically increased immune response was observed in
larvae that had been injected with 10 ␮g RNA along with heatkilled 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. 1A). 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).
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 ␮g 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/␮l) when compared with animals injected with
bacteria along with PBS (hemocyte count of 1.26 ⫾ 0.34 ⫻ 103
cells/␮l) (Fig. 1B). 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/␮l). RNA
injection alone resulted in no significant cellular immune activa-
tion (hemocyte count of 3.9 ⫾ 0.7 ⫻ 103 cells/␮l) when compared
with PBS injection (hemocyte count of 4 ⫾ 0.57 ⫻ 103 cells/␮l)
(Fig. 1B).
Moreover, high amounts of RNA (more than 50 ␮g 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
FIGURE 2. The presence of nucleic acids induces the formation of netlike 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 ␮l 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 ␮m.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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 ␮g 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 ␮g RNA were
coinjected with heat-killed P. luminescens bacteria (0.78 ⫾ 0.18 ⫻ 103 hemocytes/␮l) when compared with injected bacteria alone (1.26 ⫾ 0.34 ⫻ 103
hemocytes/␮l). 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/␮l). RNA injection alone resulted in no significant cellular immune activation (3.9 ⫾ 0.7 ⫻ 103 hemocytes/␮l) when compared with PBS
injection (4 ⫾ 0.57 ⫻ 103 hemocytes/␮l). Results represent mean values of at least three independent determinations ⫾ SD. Statistically significant
differences were determined using Student‘s t test and are indicated (ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.005).
2708
EXTRACELLULAR NUCLEIC ACIDS IN INSECT INNATE IMMUNITY
In a next step, we investigated the procoagulant potential of
nucleic acids. Larvae were pierced with a sterile needle, and hemolymph samples (⬃10 –15 ␮l) collected from the wound were
directly applied on microscope slides containing 5 ␮l 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. 2B). 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).
Identification of apolipoproteins as potential mediators of
extracellular RNA-mediated immune responses
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.
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 ␤ 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 SDSstable complexes with ApoLp-III in the Galleria hemolymph with
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 ␮g/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 ␮g/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.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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.
The Journal of Immunology
Discovery of intrinsic extracellular nucleic acids in insect
hemolymph clotting reactions
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 ␮l) collected from the wound site of
larvae on microscope slides containing 5 ␮l 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. 5G).
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 ␮m.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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.
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 ␮g/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. 4A, lanes 1 and 2).
Comparable aggregate formation was found in the presence of 20
␮g/ml RNA or DNA (Fig. 4A, lanes 3 and 5). As expected, presence of RNase or DNase, respectively, prevented enhanced aggregate formation (Fig. 4A, 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).
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. 4B). 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.
2709
2710
EXTRACELLULAR NUCLEIC ACIDS IN INSECT INNATE IMMUNITY
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
(E). Injection of 10 ␮g RNA along with P. luminescens significantly prolonged survival of infected Galleria larvae up to 35 to 42 h (F). 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.
Beneficial role of extracellular nucleic acids in the in vivo
immune defense of Galleria against Photorhabdus infection
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 ␮g 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. 6A). 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 ␮g RNA was only marginally reduced with a
50% mortality at ⬃18 h vs 50% mortality at ⬃16 h without RNA
(Fig. 6B). A further control group injected with heat-killed bacteria
resulted in 100% survival, indicating that injection injury itself did
not cause mortality.
Discussion
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 lifethreatening 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
FIGURE 7. Schematic overview of comparable roles
of extracellular nucleic acids in insects and mammals.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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. 5H). To identify potential NETs formed by insect
cells, we incubated hemolymph samples (10 ␮l) mixed with
Schneider’s insect medium (200 ␮l) (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).
The Journal of Immunology
Acknowledgments
We thank Meike Fischer for excellent technical assistance, Katja Altincicek for critical reading of the manuscript, and Monica Linder for MALDITOF-MS analysis.
Disclosures
The authors have no financial conflict of interest.
References
1. Rotem, Z., R. A. Cox, and A. Isaacs. 1963. Inhibition of virus multiplication by
foreign nucleic acid. Nature 197: 564 –566.
2. Jensen, K. E., A. L. Neal, R. E. Owens, and J. Warren. 1963. Interferon responses
of chick embryo fibroblasts to nucleic acids and related compounds. Nature 200:
433– 434.
3. Ishii, K. J., K. Suzuki, C. Coban, F. Takeshita, Y. Itoh, H. Matoba, L. D. Kohn,
and D. M. Klinman. 2001. Genomic DNA released by dying cells induces the
maturation of APCs. J. Immunol. 167: 2602–2607.
4. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate
immunity. Cell 124: 783– 801.
5. Karin, M., T. Lawrence, and V. Nizet. 2006. Innate immunity gone awry: linking
microbial infections to chronic inflammation and cancer. Cell 124: 823– 835.
6. Ishii, K. J., and S. Akira. 2007. Innate immune recognition of, and regulation by,
DNA. Trends Immunol. 27: 525–532.
7. Chi, H., and R. A. Flavell. 2007. Immunology: sensing the enemy within. Nature
448: 423– 424.
8. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D. S. Weiss,
Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps kill bacteria. Science 303: 1532–1535.
9. Brinkmann, V., and A. Zychlinsky. 2007. Beneficial suicide: why neutrophils die
to make NETs. Nat. Rev. Microbiol. 5: 577–582.
10. Urban, C. F., U. Reichard, V. Brinkmann, and A. Zychlinsky. 2006. Neutrophil
extracellular traps capture and kill Candida albicans yeast and hyphal forms.
Cell. Microbiol. 8: 668 – 676.
11. Beiter, K., F. Wartha, B. Albiger, S. Normark, A. Zychlinsky, and
B. Henriques-Normark. 2006. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr. Biol. 16: 401– 407.
12. Buchanan, J. T., A. J. Simpson, R. K. Aziz, G. Y. Liu, S. A. Kristian, M. Kotb,
J. Feramisco, and V. Nizet. 2006. DNase expression allows the pathogen group
A Streptococcus to escape killing in neutrophil extracellular traps. Curr. Biol. 16:
396 – 400.
13. Walker, M. J., A. Hollands, M. L. Sanderson-Smith, J. N. Cole, J. K. Kirk,
A. Henningham, J. D. McArthur, K. Dinkla, R. K. Aziz, R. G. Kansal, et al. 2007.
DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat. Med. 13: 981–985.
14. Sumby, P., K. D. Barbian, D. J. Gardner, A. R. Whitney, D. M. Welty,
R. D. Long, J. R. Bailey, M. J. Parnell, N. P. Hoe, G. G. Adams, et al. 2005.
Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc. Natl. Acad.
Sci. USA 102: 1679 –1684.
15. Kannemeier, C., A. Shibamiya, F. Nakazawa, H. Trusheim, C. Ruppert,
P. Markart, Y. Song, E. Tzima, E. Kennerknecht, M. Niepmann, et al. 2007.
Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation. Proc. Natl. Acad. Sci. USA 104: 6388 – 6393.
16. Pedicord, D. L., D. Seiffert, and Y. Blat. 2007. Feedback activation of factor XI
by thrombin does not occur in plasma. Proc. Natl. Acad. Sci. USA 104:
12855–12860.
17. Aperis, G., B. B. Fuchs, C. A. Anderson, J. E. Warner, S. B. Calderwood, and
E. Mylonakis. 2007. Galleria mellonella as a model host to study infection by the
Francisella tularensis live vaccine strain. Microbes Infect. 9: 729 –734.
18. Mylonakis, E., R. Moreno, J. B. El Khoury, A. Idnurm, J. Heitman,
S. B. Calderwood, F. M. Ausubel, and A. Diener. 2005. Galleria mellonella as a
model system to study Cryptococcus neoformans pathogenesis. Infect. Immun.
73: 3842–3850.
19. Jander, G., L. G. Rahme, and F. M. Ausubel. 2000. Positive correlation between
virulence of Pseudomonas aeruginosa mutants in mice and insects. J. Bacteriol.
182: 3843–3845.
20. Miyata, S., M. Casey, D. W. Frank, F. M. Ausubel, and E. Drenkard. 2003. Use
of the Galleria mellonella caterpillar as a model host to study the role of the type
III secretion system in Pseudomonas aeruginosa pathogenesis. Infect. Immun. 71:
2404 –2413.
21. Scully, L. R., and M. J. Bidochka. 2005. Serial passage of the opportunistic
pathogen Aspergillus flavus through an insect host yields decreased saprobic capacity. Can. J. Microbiol. 51: 185–189.
22. Bergin, D., L. Murphy, J. Keenan, M. Clynes, and K. Kavanagh. 2006. Preexposure to yeast protects larvae of Galleria mellonella from a subsequent lethal
infection by Candida albicans and is mediated by the increased expression of
antimicrobial peptides. Microbes Infect. 8: 2105–2112.
23. Park, S. Y., K. M. Kim, J. H. Lee, S. J. Seo, and I. H. Lee. 2007. Extracellular
gelatinase of Enterococcus faecalis destroys a defense system in insect hemolymph and human serum. Infect. Immun. 75: 1861–1869.
24. Fedhila, S., N. Daou, D. Lereclus, and C. Nielsen-LeRoux. 2006. Identification of
Bacillus cereus internalin and other candidate virulence genes specifically induced during oral infection in insects. Mol. Microbiol. 62: 339 –355.
25. Morton, D. B., G. B. Dunphy, J. S. Chadwick. 1987. Reactions of hemocytes of
immune and non-immune Galleria mellonella larvae to Proteus mirabilis. Dev.
Comp. Immunol. 11: 47–55.
26. Rowley, A. F., and N. A. Ratcliffe. 1976. The granular cells of Galleria mellonella during clotting and phagocytic reactions in vitro. Tissue Cell 8: 437– 446.
27. Bidla, G., M. Lindgren, U. Theopold, and M. S. Dushay. 2005. Hemolymph
coagulation and phenoloxidase in Drosophila larvae. Dev. Comp. Immunol. 29:
669 – 679.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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 –52) but have more similarities to mammalian fibrinplatelet 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 ␤ 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.
2711
2712
EXTRACELLULAR NUCLEIC ACIDS IN INSECT INNATE IMMUNITY
43. Halwani, A. E., D. F. Niven, and G. B. Dunphy. 2001. Apolipophorin-III in the
greater wax moth. Galleria mellonella (Lepidoptera: Pyralidae). Arch. Insect Biochem. Physiol. 48: 135–143.
44. Ma, G., D. Hay, D. Li, S. Asgari, and O. Schmidt. 2006. Recognition and inactivation of LPS by lipophorin particles. Dev. Comp. Immunol. 30: 619 – 626.
45. Theopold, U., O. Schmidt, K. Söderhäll, and M. S. Dushay. 2004. Coagulation in
arthropods: defense, wound closure and healing. Trends Immunol. 25: 289 –294.
46. Ruggeri, Z. M. 2007. Von Willebrand factor: looking back and looking forward.
Thromb. Haemost. 98: 55– 62.
47. Wood, W., and A. Jacinto. 2007. Drosophila melanogaster embryonic haemocytes: masters of multitasking. Nat. Rev. Mol. Cell Biol. 8: 542–551.
48. Smith, A. F., L. M. Owen, L. M. Strobel, H. Chen, M. R. Kanost, E. Hanneman,
and M. A. Wells. 1994. Exchangeable apolipoproteins of insects share a common
structural motif. J. Lipid Res. 35: 1976 –1984.
49. Karlsson, C., A. M. Korayem, C. Scherfer, O. Loseva, M. S. Dushay, and
U. Theopold. 2004. Proteomic analysis of the Drosophila larval hemolymph clot.
J. Biol. Chem. 279: 52033–52041.
50. Agianian, B., C. Lesch, O. Loseva, and M. S. Dushay. 2007. Preliminary characterization of hemolymph coagulation in Anopheles gambiae larvae. Dev. Comp.
Immunol. 31: 879 – 888.
51. Scherfer, C., C. Karlsson, O. Loseva, G. Bidla, A. Goto, J. Havemann,
M. S. Dushay, and U. Theopold. 2004. Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method. Curr.
Biol. 14: 625– 629.
52. Haine, E. R., J. Rolff, and M. T. Siva-Jothy. 2007. Functional consequences of
blood clotting in insects. Dev. Comp. Immunol. 31: 456 – 464.
53. Lande, R., J. Gregorio, V. Facchinetti, B. Chatterjee, Y. H. Wang, B. Homey,
W. Cao, Y. H. Wang, B. Su, F. O. Nestle, T. Zal, et al. 2007. Plasmacytoid
dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:
564 –569.
54. Vlassov, V. V., P. P. Laktionov, and E. Y. Rykova. 2007. Extracellular nucleic
acids. Bioessays 29: 654 – 667.
55. Fleischhacker, M., and B. Schmidt. 2007. Circulating nucleic acids (CNAs) and
cancer: a survey. Biochim. Biophys. Acta 1775: 181–232.
56. Kawane, K., M. Ohtani, K. Miwa, T. Kizawa, Y. Kanbara, Y. Yoshioka,
H. Yoshikawa, and S. Nagata. 2006. Chronic polyarthritis caused by mammalian
DNA that escapes from degradation in macrophages. Nature 443: 998 –1002.
57. Feingold, K. R., J. L. Funk, A. H. Moser, J. K. Shigenaga, J. H. Rapp, and
C. Grunfeld. 1995. Role for circulating lipoproteins in protection from endotoxin
toxicity. Infect. Immun. 63: 2041–2046.
58. Cole, K. D., G. P. Fernando-Warnakulasuriya, M. S. Boguski, M. Freeman,
J. I. Gordon, W. A. Clark, J. H. Law, and M. A. Wells. 1987. Primary structure
and comparative sequence analysis of an insect apolipoprotein: apolipophorin-III
from Manduca sexta. J. Biol. Chem. 262: 11794 –11800.
59. Avarre, J. C., E. Lubzens, and P. J. Babin. 2007. Apolipocrustacein, formerly
vitellogenin, is the major egg yolk precursor protein in decapod crustaceans and
is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B. BMC
Evol. Biol. 7: 3.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
28. Li, D., C. Scherfer, A. M. Korayem, Z. Zhao, O. Schmidt, and U. Theopold.
2002. Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade. Insect Biochem. Mol.
Biol. 32: 919 –928.
29. Korayem, A. M., T. Hauling, C. Lesch, M. Fabbri, M. Lindgren, O. Loseva,
O. Schmidt, M. S. Dushay, and U. Theopold. 2007. Evidence for an immune
function of lepidopteran silk proteins. Biochem. Biophys. Res. Commun. 352:
317–322.
30. Altincicek, B., and A. Vilcinskas. 2006. Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella. Dev. Comp. Immunol. 30: 1108 –1118.
31. Griesch, J., M. Wedde, and A. Vilcinskas. 2000. Recognition and regulation of
metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses. Insect. Biochem. Mol.
Biol. 30: 461– 472.
32. Altincicek, B., M. Linder, D. Linder, K. T. Preissner, and A. Vilcinskas. 2007.
Microbial metalloproteinases mediate sensing of invading pathogens and activate
innate immune responses in the lepidopteran model host Galleria mellonella.
Infect. Immun. 75: 175–183.
33. Altincicek, B., and A. Vilcinskas. 2008. Identification of a lepidopteran matrix
metalloproteinase with dual roles in metamorphosis and innate immunity. Dev.
Comp. Immunol. 32: 400 – 409.
34. Matzinger, P. 2002. The danger model: a renewed sense of self. Science 296:
301–305.
35. Jiravanichpaisal, P., B. L. Lee, and K. Söderhäll. 2006. Cell-mediated immunity
in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 211: 213–236.
36. Strand, M. R. 2008. The insect cellular immune response. Insect Science 15:
1–14.
37. Ausubel, F. M., R. Brent, R. Kingston, D. Moore, J. Seidman, J. Smith, and
K. Struhl. 1987. Current Protocols in Molecular Biology, New York: John Wiley
and Sons.
38. Boman, H. G., I. Nilsson-Faye, K. Paul, and T. J. Rasmusen. 1974. Insect immunity I: characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia. Infect. Immun. 10: 136 –145.
39. Altincicek, B., and A. Vilcinskas. 2007. Analysis of the immune-inducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly
Eristalis tenax. BMC Genomics 8: 326.
40. Ratcliffe, N. A., and S. J. Gagen. 1977. Studies on the in vivo cellular reactions
of insects: an ultrastructural analysis of nodule formation in Galleria mellonella.
Tissue Cell 9: 73– 85.
41. Wiesner, A., S. Losen, P. Kopácek, C. Weise, and P. Götz. 1997. Isolated apolipophorin III from Galleria mellonella stimulates the immune reactions of this
insect. J. Insect Physiol. 43: 383–391.
42. Whitten, M. M., I. F. Tew, B. L. Lee, and N. A. Ratcliffe. 2004. A novel role for
an insect apolipoprotein (apolipophorin III) in ␤-1,3-glucan pattern recognition
and cellular encapsulation reactions. J. Immunol. 172: 2177–2185.