Research Article
627
Drosophila Rel proteins are central regulators of a
robust, multi-organ immune network
Nina Matova* and Kathryn V. Anderson
Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
[email protected]; [email protected]
*Present address: Instituto de Medicina Molecular, Edifício Egas Moniz, 1649-028 Lisbon, Portugal
Journal of Cell Science
Accepted 21 November 2009
Journal of Cell Science 123, 627-633
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.060731
Summary
Survival of all animals depends on effective protection against infection. In Drosophila, opportunistic infection kills larvae if they lack
the Rel/NF-B proteins Dorsal and Dif. We have used tissue-specific expression of Dif and Dorsal to reveal that these Rel proteins
act in three different tissues to defend larvae from infection. Dif and Dorsal act in circulating blood cells, where they are required
autonomously to promote blood-cell survival and phagocytosis of microorganisms. We show that a major transcriptional target of
Dorsal and Dif in blood cells is Drosophila IAP1, a gene protecting these cells from death. We find that in addition to their autonomous
role in blood-cell survival, Dif and Dorsal also act in the fat body to produce factors that promote blood-cell viability. These Rel
proteins act in the epidermis to prevent infection by maintaining a barrier to microbial entry. Dorsal or Dif in any one of the three
tissues is sufficient to defend the animal from opportunistic infection. Thus Drosophila has a multi-pronged system of defense and
each branch of this network requires Rel proteins. Based on similarities between Drosophila and mammals, we propose that a Reldependent network is an ancient and robust framework of animal immune systems.
Key words: Drosophila larva, Rel protein, Hemocyte, Innate immunity
Introduction
Mammals, insects and plants all use multiple, parallel strategies to
avoid infection by the thousands of potential pathogens they
encounter daily. In mammals, the mechanisms that defend against
infection include anatomical and physiological barriers, as well as
specialized immune tissues and immune cells. This complex group
of organs and cells constitutes the body’s natural defense and it is
termed the immune system. Within the immune system, specialized
immune cells such as circulating hematopoietic cells recognize and
kill pathogens. Hematopoietic, epithelial and hepatic cells secrete
humoral components that inhibit microbial growth and attract
immune effector cells to the sites of infection. The components of
the mammalian immune system define an interactive, mutually
supportive and tightly regulated network that provides robust host
defense. Although multiple tissues participate in the execution of
the immune response, they all depend on signaling through the
Rel/NF-B family of transcription factors (Pasparakis et al., 2006;
Schmidt-Ullrich et al., 2001). Thus, Rel proteins represent the
molecular lynchpin in the mammalian host defense (Caamano and
Hunter, 2002; Hayden et al., 2006).
In Drosophila, as in mammals, several tissues protect the animal
from infection. In the larva, hemocytes (also called blood cells) have
a central role in the defense against microbial infection (Braun et al.,
1998; Defaye et al., 2009; Matova and Anderson, 2006). The vast
majority of the hemocytes are phagocytic cells that rapidly and
effectively remove microbes from circulation. The systemic humoral
response in Drosophila is mediated primarily by the fat body (the
liver analogue), which secretes antimicrobial peptides into the
hemolymph in response to infection in both the larva and the adult
(Lemaitre and Hoffmann, 2007). In addition, epithelial cells can
express antimicrobial peptide genes as a local response to infection
(Ferrandon et al., 1998; Onfelt Tingvall et al., 2001; Tzou et al., 2000).
Three Rel proteins, Dorsal-related immunity factor (Dif), Dorsal
(Dl) and Relish, play central roles in the regulation of the Drosophila
immune response. Relish is required for the production of most of
the antimicrobial peptides in the larvae, but the cellular immune
response appears to be normal in Relish mutants (Choe, 2002;
Hedengren et al., 1999; Lu et al., 2001). Dif and dorsal (dl) single
mutant larvae do not show defects in either the humoral or the cellular
immune response. However, Dif dl double mutant hemocytes have
a high rate of cell death and are unable to clear ingested microbes
(Matova and Anderson, 2006). As a consequence of hemocyte death
and defective cellular immune response, Dif dl double mutant
animals die as larvae as a result of opportunistic infection.
Although multiple tissues in Drosophila participate in the defense
against pathogens, it is not clear whether immune responses are
coordinated among different tissues and whether these tissues form
an interdependent network that could be called an immune system.
Research in this area has been focused primarily on the possible
instructive role of the blood cells in regard to the immune responses
in the fat body. Blood cells appear to perform this role by producing
cytokine-like molecules that in turn promote an immune response
in the fat body. For example, blood cells secrete a JAK/STAT
pathway ligand (Unpaired3) after septic injury, and this cytokine
induces the production of stress proteins in the fat body (Agaisse
et al., 2003). Analysis of mutants has shown that blood cells are
required for the normal induction of the antimicrobial peptide gene
Defensin in the fat body after septic injury (Brennan et al., 2007)
and for the induction of several antimicrobial-peptide genes after
natural infection (Basset et al., 2000; Dijkers and O’Farrell, 2007).
However, other possible interactions between immune-responsive
tissues have not been investigated.
In this study, we used tissue-specific expression to test whether
there are coordinated immune interactions among three different
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Journal of Cell Science 123 (4)
immune tissues in the Drosophila larva. Previously we showed Dif
and Dl act in circulating blood cells to promote their survival and
to prevent opportunistic infection (Matova and Anderson, 2006).
Here, we establish that a key target of these Rel proteins in
hemocytes is the Drosophila IAP1 (diap1; also known as thread,
th), a gene with a crucial role for cell survival in Drosophila
(reviewed by Steller, 2008). We also demonstrate that Dl and Dif
play a role in host defense in two other larval tissues. We show that
expression of Dif and Dl in the larval fat body is important for host
defense through regulation of blood-cell number. We also find that
Dl acts in the epidermis, where it prevents infection by maintaining
the proper barrier to the environment. Thus, Rel proteins are active
in these three distinct immune tissues and control a coordinated
network that mediates host defense and maintains immune
homeostasis in Drosophila.
Results
Journal of Cell Science
Autonomous Dif or Dl activity promotes blood-cell
survival through positive regulation of diap1
Previously we showed that more than 95% of Dif dl double mutant
animals die during larval stages due to opportunistic infection by
bacteria and fungi from the environment (Matova and Anderson,
2006) (Table 1). Defects in blood cells are a primary cause of
infection and death in these animals: blood-cell number is reduced
due to a high rate of cell death, and the surviving blood cells are
abnormal in morphology and fail to phagocytose microorganisms
(Matova and Anderson, 2006) (Fig. 1B,C). We used the UAS/GAL4
system to perform tissue-specific rescue experiments and showed
that expression of either Dif or dl in Dif dl hemocytes rescues
hemocyte number, morphology and function, prevents infection and
allows survival to adulthood (Matova and Anderson, 2006). For
example, when Peroxidasin-Gal4, a line that directs expression in
the majority of the hemocytes but not in any other tissues (Stramer
et al., 2005) (Materials and Methods) was used to express either
Dif or dl in double mutant larvae, approximately 50% of Dif dl
animals that carried UAS-Dif or UAS-dl survived to adulthood (Table
1). As observed with other blood-cell drivers (Matova and Anderson,
2006), hemolymph samples from these larvae were microbe-free,
the morphology of the blood cells was normal (Fig. 1D) and
hemocyte numbers approached the number in wild-type larvae
(Table 1).
There are no known transcriptional targets of Dl or Dif in blood
cells. Previously we have shown that Dl and Dif are required for
blood-cell survival (Matova and Anderson, 2006): 15% of the
hemocytes in third instar Dif dl larvae are TUNEL-positive,
compared to 2.3% of hemocytes in wild-type larvae. Because of
the potent anti-apoptotic activities of Dl and Dif and because diap1
is the principal negative regulator of apoptosis in somatic cells
(Steller, 2008), we investigated whether Dif and Dl regulate diap1.
We first tested whether constitutive expression of diap1 in Dif dl
mutants could modify the Dif dl mutant phenotype. To that end,
we expressed UAS-diap1 in the hemocyte lineage of Dif dl mutants
using a Hemese (He)-Gal4 driver. 38.5% of these animals survived
to adulthood compared with 6.7% of Dif dl animals that carried
only one transgene (He-Gal4 without UAS-diap1). Hemolymph
samples from Dif dl animals that expressed diap1 under the control
of He-Gal4 showed that the hemocyte morphology was similar to
that of wild-type hemocytes (Fig. 1E). The rescued larvae had only
occasional microbes and their hemocyte numbers were four times
higher than unrescued Dif dl animals (Table 1). Thus constitutive
expression of diap1 in Dif dl mutants could rescue all phenotypes
of Dif dl animals.
To test whether Dl and Dif are required for normal expression
of DIAP1, we assayed DIAP1 protein levels in wild-type and Dif
dl hemocytes. Staining with DIAP1 antibody (Ryoo et al., 2002)
showed predominantly cytoplasmic distribution of DIAP1 in wildtype hemocytes (Fig. 2A). DIAP1 protein was severely reduced or
undetectable in Dif dl hemocytes (Fig. 2B). By contrast, there was
a robust DIAP1 expression in the fat body of Dif dl larvae (not
shown). Thus, DIAP1 is specifically downregulated in Dif dl blood
cells.
To test whether diap1 is a transcriptional target of the Rel proteins
in blood cells we employed a diap1 enhancer trap, thj5c8 P{lacZ},
that has been used previously to monitor diap1 transcription in the
Table 1. Blood cell number and survival in Dif dorsal double mutants
Genotype
UAS line
Gal4 line
Wild type
Dif dl
Dif dl
None
None
UASp-dl
Dif dl
UAS-Dif
Dif dl
UAS-diap1
Dif dl
UAS-Defensin
None
None
hs-Gal4
Pxn-Gal4
Lsp2-Gal4
e13C-Gal4
hs-Gal4
Pxn-Gal4
Lsp2-Gal4
He-Gal4
Lsp2-Gal4
Lsp2-Gal4
He-Gal4
Target tissue
Hemocyte
number/l (⫻103)
Adult survival
(% of expected)
Infection
status
Ubiquitous
BC, LG
Fat body
Epidermis
Ubiquitous
BC, LG
Fat body
BC
Fat body
Fat body
BC
7.2±1.7
0.9±0.5
ND
6.4±4.7
4.2±2.0
1.0±0.2
ND
8.5±4.6
4.4±1.6
3.6±2.6
0.7±0.6
1.2±0.8
1.2±0.6
100
4.4
100
54
40
10
130
50
45
39
7.6
8
8
–
+++
ND
–
+
–
ND
–
+
+
+++
+++
+++
Hemocyte number is the average cell count from six animals of the indicated genotypes.
UASp-dl or UAS-Dif line were expressed under the control of the corresponding Gal4 driver at 25°C. Crosses that use hs-Ga4 were performed at 25°C, i.e.
without heat shock.
Adult survival was determined by counting 200-800 total progeny in each experiment and percentage survival was calculated by comparing the survival of Dif
dl mutants that carried both the UAS and Gal4 transgenes to the number of Dif dorsal/++ heterozygous siblings from the same cross (defined as 100%). The
percentage of survival is represented as the weighted average of at least ten different crosses and is thus normalized for differences in culturing conditions.
Infection status was determined by counting the number of microbes in a standard microscope field: +++, ~105 microbes per animal; +, less than 102 microbes
per animal. Wild-type hemolymph samples have no microbes.
ND, not determined; BC, blood cells; LG, lymph glands; hs-Gal4, heat shock-Gal4; Pxn-Gal4, peroxidasin-Gal4; He-Gal4, Hemese-Gal4.
Journal of Cell Science
A Drosophila Rel protein network
629
Fig. 1. Tissue-specific rescue of Dif dl hemolymph and hemocyte phenotypes. Hemolymph samples were stained with Filamin antibody (Sokol and Cooley,
1999) (red; upper panels) to reveal the F-actin, and with DAPI (lower panels) in cyan (upper panels) to show nuclear and microbial DNA. (A)Wild-type
hemocytes are either small round cells with pronounced subcortical F-actin or larger cells with numerous microspikes (arrow). Wild-type hemolymph samples
do not contain microbes. (B,C)Dif dl mutant hemocytes appear ruptured (B) or have an irregular shape and a meshwork of cytoplasmic actin filaments (C). Dif
dl hemolymph contains microbes in the absence of experimentally introduced infection (B⬘, arrow). (D)Dif dl larvae that express UASp-dl in blood cells under
the control of Pxn-Gal4 line do not have microbes in the hemolymph. Hemocytes have the same morphology as wild-type hemocytes. (E)Dif dl larvae that
express UAS-diap1 in hemocytes under the control of He-Gal4 line have only occasional microbes in the hemolymph. Hemocyte morphology is similar to the
morphology of wild-type hemocytes. (F,G)Dif dl larvae that express UASp-dl in the fat body under the control of Lsp2-Gal4 have a low level of microbial
infection in the hemolymph (F⬘, arrow). The cell shape and subcellular distribution of F-actin in 40-60% of the hemocytes is very abnormal (F). The remaining
hemocytes have morphology that is more similar to wild type (G). (H)Dif dl larvae that express UASp-dl in the epidermis under the control of e13C-Gal4 line
do not have microbes in the hemolymph. However, their hemocytes have an irregular shape and abnormal nuclei. Microbes are seen only occasionally inside
the hemocytes (arrow in H⬘). Scale bar: 10m.
wing imaginal disc (Ryoo et al., 2002). Anti--galactosidase (Gal) labeling revealed a nuclear staining in wild-type hemocytes
carrying the enhancer trap (Fig. 2C). The nuclear staining reflects
the presence of a nuclear localization signal in the P{lacZ} vector.
By contrast, in Dif dl hemocytes that carried the reporter enhancer
trap, the nuclear staining was greatly diminished (Fig. 2D). We
quantified diap1 transcription by measuring pixel intensity of
nuclear -Gal fluorescence in wild-type and Dif dl hemocytes. The
measurements showed that expression levels of diap1 were 5.8fold lower (P4.4⫻10–8, Student’s t-test) in Dif dl blood cells than
in wild-type hemocytes (supplementary material Fig. S1). Thus Dif
and Dl are positive regulators of diap1 expression in blood cells
and diap1 is a key target of Dif and Dl in the regulation of bloodcell survival.
Dif and Dl activity in the fat body can regulate hemocyte
number
To test whether the roles of Dif and Dl in immunity could be
explained exclusively by their functions in blood cells, we examined
the effect of expressing Dl or Dif in other immune-responsive
tissues. We assayed survival to adulthood, presence of infection in
hemolymph samples, hemocyte numbers and hemocyte
morphology. We first focused on the fat body, a major immune organ
in the Drosophila larva (Lemaitre and Hoffmann, 2007). To test
Fig. 2. Dif and Dl are positive regulators of diap1 expression. (A,B). Hemolymph samples were stained with DIAP1 antibody (red) and DAPI (blue). A⬘ and B⬘
show DIAP1 antibody staining only. Images of the wild-type and Dif dl hemocytes were acquired at identical settings. (A,A⬘) Wild-type hemocytes show
predominantly cytoplasmic distribution of DIAP1. (B,B⬘) Dif dl hemocytes show a significant reduction in DIAP1 protein. Scale bar: 10m. (C,D)Hemolymph
samples were stained with -Gal antibody (red) and DAPI (blue). C⬘ and D⬘ show -Gal staining only. Images of the wild-type and Dif dl hemocytes were acquired
at identical settings. (C,C⬘) Wild-type hemocytes carrying a diap1 enhancer trap, thj5c8 P{lacZ}, show a predominantly nuclear localization of -Gal . The nuclear
staining reflects the presence of a nuclear localization signal in the P{lacZ} vector. (D,D⬘) Dif dl hemocytes carrying a diap1 enhancer trap, thj5c8 P{lacZ}, show a
significant reduction in -Gal staining. The intensity of -Gal fluorescence in wild-type and Dif dl hemocytes is quantified in supplementary material Fig. S1. Scale
bar: 10m.
Journal of Cell Science
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Journal of Cell Science 123 (4)
Fig. 3. Constitutive expression of Def does not rescue Dif dl hemocyte
phenotypes. (A)Expression of Def as assayed by real-time PCR. Uninfected
wild-type larvae (genotype: Dif dl/++; Lsp2) do not express Def. 2 hours after
injection with E. coli DH5, Def expression is induced 85-fold in wild-type
larvae. Dif dl larvae expressing Def under the control of Lsp2-Gal4 (Dif dl;
Lsp2>Def) or under the control of Hemese-Gal4 (Dif dl; He>Def) express
very high levels of Def. Def expression is represented on the y-axis as the ratio
of the average Def induction to the induction of Rpl32 (AvgDef/Rpl32).
(B,C)Hemolymph samples from wild-type larvae (genotype: Dif dl/++; Lsp2)
have intact blood cells and are microbe-free (B). By contrast, hemolymph
samples of Dif dl larvae expressing Def under the control of Lsp2-Gal4 (Dif
dl; Lsp2>Def) show abundant bacteria (arrows) and ruptured hemocytes (C).
Similar results were obtained in hemolymph samples from Dif dl larvae
expressing Def under the control of Hemese-Gal4 (Dif dl; He>Def).
Hemolymph samples were stained with Filamin antibody (red) to reveal the Factin, and DAPI (cyan) to show nuclei and microbial DNA. Scale bar: 10m.
the contribution of Dif and Dl in the larval fat body, we expressed
UAS-Dif or UASp-dl in Dif dl animals using the Lsp2-Gal4 driver,
a line that drives expression only in the fat body (Cherbas et al.,
2003) (and our data). Forty to forty-five percent of the Lsp2-Gal4rescued Dif dl double mutants survived to adulthood and their
hemolymph samples had only few microbes, compared with the
rampant infection in Dif dl siblings (Fig. 1F,G, Table 1). Thus,
expression of either Dif or dl in the fat body protected the double
mutant larvae from infection.
Because blood cells play a central role in the ability of larvae to
fight infection, we examined the hemocyte number and morphology
in the fat body-rescued Dif dl animals. Expression of either Dif or
dl in the fat body increased the number of circulating hemocytes
in Dif dl mutants nearly fivefold, to about 60% of wild-type numbers
(Table 1).
As described above, 15% of the hemocytes in third instar Dif dl
larva are TUNEL-positive (Matova and Anderson, 2006). By
contrast, only 5.8% of the hemocytes in Dif dl larvae that expressed
dl in the fat body were TUNEL-positive (supplementary material
Table S1). When normalized to the total number of hemocytes
(supplementary material Table S1), the percentage of TUNELpositive cells in Dif dl larvae that expressed dl in the fat body is
nearly threefold lower than in Dif dl mutants alone. The rescued
hemocytes did not express Dl protein (supplementary material Fig.
S2), indicating that Rel proteins in the fat body acted in a nonautonomous manner to promote survival of blood cells. The
morphological abnormalities of Dif dl mutant hemocytes were not
fully rescued by the expression of the Rel genes in the fat body:
most hemocytes were irregular in shape and had a meshwork of
cytoplasmic filamentous actin, like unrescued Dif dl blood cells (Fig.
1C,F,G). These observations indicate that a fat body-derived factor
promotes survival of blood cells but cannot rescue a cellautonomous requirement for Rel proteins in maintenance of bloodcell morphology.
There are no obvious candidate genes that would encode the
factor(s) made by the fat body and protecting blood cells. Expression
of diap1 exclusively in the fat body with the Lsp2-Gal4 driver did
not rescue either blood-cell number or survival to adulthood (Table
1). Because antibiotic treatment can protect Dif dl mutant larvae
from infection (Matova and Anderson, 2006), it was possible that
the antimicrobial peptides secreted by the fat body (Lemaitre and
Hoffmann, 2007) might increase the likelihood of blood-cell
survival. Although we previously demonstrated that Dif dl mutant
larvae induce all of the major antimicrobial peptide genes (Matova
and Anderson, 2006), Defensin (Def) was the only antimicrobial
peptide whose induction after septic injury was significantly reduced
in the double mutants, and was expressed at about 30% of the levels
present in the wild type. Among the antimicrobial peptides, Def
appears to have the most potent activity: constitutive expression
of Def confers resistance to Gram-positive bacteria in
immunocompromised adults (Tzou et al., 2002) and to
Pseudomonas, a Gram-negative bacterium in wild-type adult
Drosophila (Apidianakis et al., 2005). To test whether Def could
protect Dif dl larvae from infection, we used the fat body-specific
Lsp2-Gal4 driver and the hemocyte-specific He-Gal4 driver to
express UAS-Def. Real-time PCR experiments showed that there
was twice the level of Def in Dif dl larvae that expressed Def with
the fat-body driver and five-fold more Def in double mutants that
expressed Def with the blood-cell driver, compared with infected
wild-type animals (Fig. 3A). High levels of Def expression did not
significantly increase the number of blood cells (Table 1), indicating
that Def is not the blood-cell survival factor. Expression of Def
increased the likelihood of survival to adult stages only slightly
(8% vs 4.4% survival) (Table 1) and did not prevent infection in
most animals (five out of seven animals examined were infected;
Fig. 3B). Even in those animals in which the infection load was
reduced, the morphology of the blood cells was as abnormal as in
the Dif dl mutant siblings. Thus, despite the potent activity of Def
in adult animals, overexpression of Def was not sufficient to protect
Dif dl double mutant larvae from opportunistic infection and Def
is not the Rel-dependent fat-body factor that promotes hemocyte
survival.
Rel protein activity in the epidermis protects from
infection
In addition to hemocytes and the fat body, the larval epidermis is
another immune organ that provides a mechanical barrier to
infection and also expresses antimicrobial peptides that could defend
the animal from invading microbes (Onfelt Tingvall et al., 2001).
Journal of Cell Science
A Drosophila Rel protein network
We observed that Dif dl larvae had obvious defects in the epidermis:
70% of the double mutants exhibited melanized spots of variable
size and location throughout the epidermis (Fig. 4B; supplementary
material Table S2), whereas only 5.5% of the wild-type larvae had
detectable epidermal defects (supplementary material Table S2).
These spots did not clear and persisted throughout the life of Dif
dl larvae. To look more closely at the structure of the larval epidermis
in wild-type and in Dif dl animals, we marked the epidermal cells
with GFP::actin and DAPI. This examination revealed that the
global organization of the epidermis was similar in wild-type and
Dif dl animals (Fig. 4C,D). Confocal scanning microscopy through
the melanized lesions in Dif dl larvae showed that the lesions
spanned the entire epidermal layer and did not have detectable
GFP::actin or DAPI fluorescence (Fig. 4F).
The melanized-spot phenotype of Dif dl larvae was rescued by
expression of dl in the epidermis with e13C-Gal4, a line that drives
strong expression in the epidermis but not in hemocytes or in the
fat body (supplementary material Table S2). Thus, Dl is required
autonomously in larval epidermal cells to prevent epidermal lesions.
Because expression of UAS-Dif with the e13C-Gal4 caused lethality
in both the wild-type and Dif dl mutant background, the effect of
Dif expression in the epidermis of Dif dl mutants could not be
assayed.
To test whether dl in the epidermis protects the larva from
infection, we examined hemolymph samples of Dif dl mutants that
expressed dl with the e13C-Gal4 line. Although a few bacteria were
still detectable inside hemocytes (Fig. 1H⬘, arrow), no microbes
were seen in the hemolymph of these animals. Nevertheless, the
number of circulating blood cells in these larvae remained as low
as in unrescued Dif dl double mutants (Table 1) and these blood
cells retained the abnormal morphology seen in Dif dl double mutant
larvae (Fig. 1H). Epidermal expression of dl improved the rate of
survival of Dif dl mutants to the third instar larvae, but these animals
arrested at the onset of metamorphosis and did not survive to adult
stages. We conclude that Rel protein activity in the epidermis
protects larvae from microbes in the environment without affecting
the number or the morphology of blood cells.
Discussion
In this study we show that Rel proteins in three different tissues –
the epidermis, the circulating blood cells and the fat body – protect
Drosophila larvae from microbial infection. These functions of the
Drosophila Rel proteins have close parallels to the functions of the
mammalian Rel proteins in immune-responsive tissues that form
the backbone of the vertebrate host defense.
Rel activity in blood cells and in the fat body controls
hemocyte numbers
Because circulating phagocytic cells play an essential role in the
clearance of microbial infection in the Drosophila larva,
maintenance of correct number of healthy hemocytes is crucial for
the survival of the animal. We find that two Rel proteins, Dl and
Dif, function both in hemocytes and in the fat body to promote
hemocyte survival. Even though the mechanisms of blood-cell
protection are different, these results revealed that the immuneresponsive organs in Drosophila interact and form a mutually
supportive network. This conclusion is further strengthened by
earlier findings indicating that signals from blood cells can stimulate
production of antimicrobial and stress peptides in the fat body
(Agaisse et al., 2003; Basset et al., 2000; Brennan et al., 2007;
Dijkers and O’Farrell, 2007). Thus there appear to be bidirectional
631
Fig. 4. Epidermal lesions in Dif dl larvae. (A,B)Images of wild-type larva
(A) and Dif dl larva (B). Dif dl larvae have melanized epidermal spots of
variable size (arrow in B) that are not present in wild-type animals.
(C,D)Confocal images of the epidermal cells of wild-type larvae (C) resemble
the epidermal cells of Dif dl larvae (D). Epidermal cells express GFP::actin
(green) under the control of e13-Gal4 line and are stained with DAPI (blue) to
show their nuclei. Scale bars: 10m. (E)Dif dl larva: a DIC image showing a
portion of the ventral epidermis with rows of denticles and a melanized
epidermal lesion (arrow). (F)High magnification confocal image of the lesion
shown in E. Epidermal cells expressing GFP::actin (green) under the control of
e13-Gal4 line were additionally stained with DAPI (blue). Note that the lesion
does not express GFP::actin and does not stain with DAPI. Scale bar: 10m.
signals between the fat body and the blood cells that coordinate
host defense responses in Drosophila larvae.
Our experiments identified diap1 as the first target of Dl and Dif
in Drosophila blood cells. Dif and Dl autonomously promote
hemocyte survival and diap1 is likely to be their principal target,
as hemocyte number, hemocyte morphology and survival to
adulthood of Dif dl double mutants can be rescued to nearly the
same extent by expression of diap1 in the hemocyte lineage as when
Dif or dl is expressed in those cells.
Mammalian blood cells also rely on Rel proteins to prevent
apoptosis: c-Rel RelA double mutant hematopoietic progenitors
cannot rescue lethally irradiated wild-type mouse hosts, and the
double-mutant macrophages have a high rate of apoptosis
(Grossmann et al., 1999). The parallels with mammals extend
further: just as Drosophila Rel proteins regulate the expression of
the inhibitor of apoptosis DIAP1 in blood cells, mammalian Rel
proteins control the expression of homologous molecules such as
c-IAP and XIAP (Karin and Lin, 2002).
Our analysis revealed a previously unsuspected role of the fat
body in controlling the number of blood cells. The results suggest
that Dif and Dl in the fat body protect the animal from infection
because they regulate blood-cell number and not because they are
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Journal of Cell Science 123 (4)
required for the production of antimicrobial peptides. Although it
is conceivable that a particular cocktail of Rel-controlled
antimicrobial peptides made in the fat body prevents blood-cell
death, it is unlikely, given that the peptides are expressed in Dif dl
larvae (Matova and Anderson, 2006) and that overexpression of
Def has no effect on blood-cell survival (Fig. 3). Instead, we propose
that Dl and Dif in the fat body control the production of survival
factors that are released into the hemolymph. It is known that the
fat body secretes a family of proteins that promote survival or
proliferation of imaginal disc cells in a concentration-dependent
manner (Kawamura et al., 1999) and these growth factors, or other
unidentified factors, might also be active on blood cells.
Just as Rel protein activity in the Drosophila fat body provides
factors that promote survival or proliferation of blood cells, the
mammalian liver (the analogue of the fat body) secretes growth
factors such as the hepatic growth factor (HGF) and interleukin7 that act as survival and proliferation molecules for blood cells
(Sawa et al., 2009; Sugiura et al., 2007; Yu et al., 1998), although
it is not known whether Rel proteins directly regulate their
production.
Journal of Cell Science
Distinct functions of Rel proteins in blood cells and the
epidermis protect the animal from infection
The opportunistic microbial infection in Dif dl hemolymph can be
rescued by expression of Dif or Dl in either the blood cells or in
the epidermis. Expression of dl in the epidermis of Dif dl animals
does not affect hemocyte number, morphology or ability to
phagocytose bacteria; nevertheless, dl expression in the epidermis
is sufficient to protect the animal from microbial infection. Hence,
these two tissues have different roles in host defense – the blood
cells remove microbes from circulation whereas the epidermis
prevents entry of microbes into the animal – but both rely on Rel
proteins for their immune functions.
The function of the Rel proteins in the epidermis is not clear. It
is possible that the lesions in the epidermis of Dif dl larvae are the
result of a developmental defect. Alternatively, because each animal
has only one or a few epidermal lesions and their location varies,
we prefer the hypothesis that epidermal gaps arise because of
defective healing of wounds that occur normally in the life of the
larva (Brock et al., 2008). In addition, Rel signaling in epidermal
cells might help to prevent infection by local expression of products
with antimicrobial activity (Onfelt Tingvall et al., 2001).
The epidermal function of Drosophila Rel proteins might also
parallel Rel functions in mammals, as mice that lack both RelA and
c-rel have defects in epidermal development and immune
homeostasis (Gugasyan et al., 2004). In agreement with these
findings, expression of a dominant negative IkBa transgene in the
epidermis of the mouse, which should block activity of several Rel
proteins, causes progressive skin disease and a strong local
inflammatory response (van Hogerlinden et al., 2004). However,
future experiments that involve conditional inactivation of essential
Rel family genes might provide a definitive assay for the function
of Rel proteins in the mammalian epidermis.
An evolutionarily conserved network of Rel-dependent
responses prevents infection
It is striking that, as in mammals, host defense in Drosophila
depends on Rel proteins in multiple tissues, although it is likely
that the Rel transcriptional targets are different in each tissue. These
parallels with the mammalian immune response suggest that the
action of Rel proteins in multiple tissues was selected early in
metazoan evolution as a mechanism to provide a robust immune
network that effectively prevents microbial infection. Future studies
on the tissue-specific targets of Rel proteins should define how much
of this immune network was present in the common ancestor of
vertebrates and invertebrates. Because of the remarkable
conservation in the framework of the immune system between
mammals and insects, characterization of Rel targets in Drosophila,
which has only three Rel genes and can be easily manipulated
genetically, could guide future experiments in mammals.
Materials and Methods
Drosophila stocks
Dif dl double mutants were transheterozygotes for two overlapping deficiencies
Df(2L)J4 and Df(2L)TW119, as described previously (Matova and Anderson, 2006).
Lsp2-Gal4 was obtained from the Bloomington Stock Center and the e13-Gal4 line
came from Norbert Perrimon (Harvard Medical School, Boston, MA). The UASpGFP::actin and UAS-Def transgenic flies were obtained from Andrew Hudson and
Lynn Cooley (Yale University, New Haven, CT) and Bruno Lemaitre (École
Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), respectively. The UASdiap1 line (Betz et al., 2008) and thj5c8 allele were provided by Hermann Steller
(Rockefeller University, New York, NY). UASp-dl and UAS-Dif have been described
previously (Matova and Anderson, 2006).
We determined the expression pattern of the Gal4 lines by crossing to stocks
carrying UAS-GFP. Pxn-Gal4 drove expression in approximately 70% of the blood
cells, lymph glands (first pair), in a part of the proventriculus, and in a subset of
brain cells. Lsp2-Gal4 drove expression exclusively in fat body cells (Cherbas et al.,
2003) (and our data). He-Gal4 drove expression in blood cells, lymph glands (a few
cells), midgut, salivary glands (Zettervall et al., 2004) (and our data). e13C-Gal4
drove expression in the epidermis, pharynx, salivary glands, ventral nerve cord, brain
and Malpighian tubules.
Tissue-specific expression of Dif and dl
Dif dl double mutants were obtained from the cross between
Df(2L)J4/T(2;3)CyO;TM6B and Df(2L)TW119/T(2;3)CyO;TM6B. Double mutants
were identified by the absence of the Tubby marker carried by the CyO;TM6B
translocation. The CyO;TM6B balancer translocation was also used to identify Dif
dl larvae that expressed Dif or dl in a tissue-specific manner. In these rescue
experiments, the UASp-dl transgene was recombined onto the Df(2L)J4 chromosome
or a third chromosome insertion of UAS-Dif line was used. All Gal4 lines used in
the experiments mapped to the third chromosome. Dif dl larvae that carried both the
UAS and Gal4 transgenes were the non-Tubby larvae from crosses from two doubly
balanced lines: e.g. Df(2L)J4 UASp-dl/T(2;3)CyO;TM6B ⫻ Df(2L)TW119; e13CGal4/T(2;3)CyO;TM6B.
For each genotype, hemolymph from three to eight non-Tubby larvae was analyzed.
Expression of UAS-Dif with the e13C-Gal4 caused lethality in both the wild-type
and Dif dl mutant backgrounds. Because of that, the effect of Dif expression in the
epidermis of Dif dl mutants could not be assayed.
Immunocytochemistry and microscopy
Blood cells samples were collected, stained and imaged as described previously
(Matova and Anderson, 2006). Hemocyte counts, infection and TUNEL were
performed as described previously (Matova and Anderson, 2006). Staining for -Gal
was performed with a rabbit antibody from Cappel, which was used at a dilution of
1:2200. DIAP1 stainings were performed either with the rabbit anti-DIAP1 serum
(diluted at 1:2000) or with the affinity-purified DIAP1 antibody (diluted at 1:300).
Both preparations produced similar results. The stainings with -Gal and DIAP1
antibody were performed in animals reared on apple-juice agar plates, which had a
lower level of hemolymph infection than the animals grown on standard cornmealmolasses-yeast medium.
The epidermis of Dif dl mutants and Dif dl/++ siblings expressing GFP::actin under
the control of e13C-Gal4 was dissected and fixed for 20 minutes in 4%
paraformaldehyde. The epidermal samples were subsequently washed in PBT and
stained with DAPI (4 mg/ml) for 2 hours at room temperature. Samples were washed
again and mounted in Vectashield (Vector laboratories) for examination. Images of
the epidermis were acquired with an inverted Leica TCS SP2 confocal microscope
using a 20⫻ 0.7 NA lens.
Analysis of Def expression
Infected wild-type animals were obtained by injecting third-instar larvae with E. coli
DH5. These larvae were homogenized 2 hours later and RNA was prepared using
RNA STAT-60 (TEL-TEST). For all other genotypes, uninfected third instar larvae
were homogenized and RNA was prepared using RNA STAT-60. Q-PCR was
performed with pre-designed TaqMan Gene expression assays for Def
(Dm01818074_s1) and Ribosomal protein L32 (Dm02151827_s1) as described
previously (Matova and Anderson, 2006). Fold changes reported are the average value
of triplicate experiments.
A Drosophila Rel protein network
Quantification of diap1 expression levels
The levels of diap1 transcription were determined by crossing the diap1 enhancer
trap, thj5c8 P{lacZ}, into wild-type and Dif dl animals. Hemocytes were stained with
-Gal antibody and DAPI. Fluorescence images were captured with a Zeiss Axiovert
200M microscope equipped with a CoolSNAP HQ CCD (Roper Scientific
Photometrics) and the MetaMorph software (Universal Imaging Corporation). All
hemocyte nuclei were identified by DAPI staining. Fluorescence intensity of nuclear
-Gal was measured in arbitrary units in 50 individual nuclei using the ImageJ 1.410
software package (Particle analysis plug-in).
Statistical analysis
Journal of Cell Science
Hemocyte numbers in Table 1 were compared using unpaired two-tailed Student’s
t-test. When compared with Dif dl larvae, hemocyte numbers were significantly higher
in Dif dl mutants that expressed UASp-dl with Pxn-Gal4, UAS-Dif with Pxn-Gal4,
UASp-dl with Lsp2-Gal4, UAS-Dif with Lsp2-Gal4 or UAS-diap1 with He-Gal4
(Student’s t-test, P<0.05).
When compared with Dif dl larvae, hemocyte numbers were not significantly higher
in Dif dl mutants that expressed UASp-dl with e13C-Gal4, UAS-Def with Lsp2-Gal4,
UAS-Def with He-Gal4 or UAS-diap1 with Lsp2-Gal4 (Student’s t-test, P>0.05).
Percentages of TUNEL-positive hemocytes in different genetic backgrounds were
compared using a Chi-square test in supplementary material Table S1. Chi-square
test was also used to compare animals with epidermal lesions versus animals without
lesions in supplementary material Table S2.
We are grateful to Katia Manova and the Molecular Cytology Facility
and to Agnes Viale and the Genomics Core Facility for experimental
support. We thank Andrew Hudson and Lynn Cooley for the UASpGFP::actin flies, Bruno Lemaitre for the UAS-Def line, Hermann Steller
and Joe Rodriguez for DIAP1 reagents, Hyung Don Ryoo for the
affinity-purified polyclonal DIAP1 antibody, and Céline Carret,
Polloneal Ocbina and José Rino for the help with statistics and the
quantification of diap1. We thank J. Bloomekatz, J. Lee, I. Migeotte
and S. Weatherbee for comments on the manuscript. This work was
supported by NIH grant AI45149 to K.V.A. and the Lita Annenberg
Hazen Foundation. N.M. was a recipient of a fellowship from the
Irvington Institute for Immunological Research and is currently
supported by Fundação para a Ciência e Tecnologia (FCT) of the
Portuguese Ministry of Science. Deposited in PMC for release after 12
months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/4/627/DC1
References
Agaisse, H., Petersen, U. M., Boutros, M., Mathey-Prevot, B. and Perrimon, N. (2003).
Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury.
Dev. Cell 5, 441-450.
Apidianakis, Y., Mindrinos, M. N., Xiao, W., Lau, G. W., Baldini, R. L., Davis, R. W.
and Rahme, L. G. (2005). Profiling early infection responses: Pseudomonas aeruginosa
eludes host defenses by suppressing antimicrobial peptide gene expression. Proc. Natl.
Acad. Sci. USA 102, 2573-2578.
Basset, A., Khush, R. S., Braun, A., Gardan, L., Boccard, F., Hoffmann, J. A. and
Lemaitre, B. (2000). The phytopathogenic bacteria Erwinia carotovora infects
Drosophila and activates an immune response. Proc. Natl. Acad. Sci. USA 97, 33763381.
Betz, A., Ryoo, H. D., Steller, H. and Darnell, J. E., Jr (2008). STAT92E is a positive
regulator of Drosophila inhibitor of apoptosis 1 (DIAP/1) and protects against radiationinduced apoptosis. Proc. Natl. Acad. Sci. USA 105, 13805-13810.
Braun, A., Hoffmann, J. A. and Meister, M. (1998). Analysis of the Drosophila host
defense in domino mutant larvae, which are devoid of hemocytes. Proc. Natl. Acad. Sci.
USA 95, 14337-14342.
Brennan, C. A., Delaney, J. R., Schneider, D. S. and Anderson, K. V. (2007). Psidin is
required in Drosophila blood cells for both phagocytic degradation and immune activation
of the fat body. Curr. Biol. 17, 67-72.
Brock, A. R., Babcock, D. T. and Galko, M. J. (2008). Active cop, passive cop:
developmental stage-specific modes of wound-induced blood cell recruitment in
Drosophila. Fly (Austin) 2, 303-305.
Caamano, J. and Hunter, C. A. (2002). NF-B family of transcription factors: central
regulators of innate and adaptive immune functions. Clin. Microbiol. Rev. 15, 414-429.
Cherbas, L., Hu, X., Zhimulev, I., Belyaeva, E. and Cherbas, P. (2003). EcR isoforms
in Drosophila: testing tissue-specific requirements by targeted blockade and rescue.
Development 130, 271-284.
633
Choe, K.-M. (2002). Molecular Characterization of Drosophila Immunity Mutants. New
York: Cornell University.
Defaye, A., Evans, I., Crozatier, M., Wood, W., Lemaitre, B. and Leulier, F. (2009).
Genetic ablation of Drosophila phagocytes reveals their contribution to both development
and resistance to bacterial infection. J. Innate Immunity 1, 322-334.
Dijkers, P. F. and O’Farrell, P. H. (2007). Drosophila calcineurin promotes induction of
innate immune responses. Curr. Biol. 17, 2087-2093.
Ferrandon, D., Jung, A. C., Criqui, M., Lemaitre, B., Uttenweiler-Joseph, S., Michaut,
L., Reichhart, J. and Hoffmann, J. A. (1998). A drosomycin-GFP reporter transgene
reveals a local immune response in Drosophila that is not dependent on the Toll pathway.
EMBO J. 17, 1217-1227.
Grossmann, M., Metcalf, D., Merryfull, J., Beg, A., Baltimore, D. and Gerondakis, S.
(1999). The combined absence of the transcription factors Rel and RelA leads to multiple
hemopoietic cell defects. Proc. Natl. Acad. Sci. USA 96, 11848-11853.
Gugasyan, R., Voss, A., Varigos, G., Thomas, T., Grumont, R. J., Kaur, P., Grigoriadis,
G. and Gerondakis, S. (2004). The transcription factors c-rel and RelA control epidermal
development and homeostasis in embryonic and adult skin via distinct mechanisms.
Mol. Cell. Biol. 24, 5733-5745.
Hayden, M. S., West, A. P. and Ghosh, S. (2006). NF-B and the immune response.
Oncogene 25, 6758-6780.
Hedengren, M., Asling, B., Dushay, M. S., Ando, I., Ekengren, S., Wihlborg, M. and
Hultmark, D. (1999). Relish, a central factor in the control of humoral but not cellular
immunity in Drosophila. Mol. Cell 4, 827-837.
Karin, M. and Lin, A. (2002). NF-B at the crossroads of life and death. Nat. Immunol.
3, 221-227.
Kawamura, K., Shibata, T., Saget, O., Peel, D. and Bryant, P. J. (1999). A new family
of growth factors produced by the fat body and active on Drosophila imaginal disc cells.
Development 126, 211-219.
Lemaitre, B. and Hoffmann, J. (2007). The host defense of Drosophila melanogaster.
Annu. Rev. Immunol. 25, 697-743.
Lu, Y., Wu, L. P. and Anderson, K. V. (2001). The antibacterial arm of the Drosophila
innate immune response requires an IB kinase. Genes Dev. 15, 104-110.
Matova, N. and Anderson, K. V. (2006). Rel/NF-B double mutants reveal that cellular
immunity is central to Drosophila host defense. Proc. Natl. Acad. Sci. USA 103, 1642416429.
Onfelt Tingvall, T., Roos, E. and Engstrom, Y. (2001). The imd gene is required for local
Cecropin expression in Drosophila barrier epithelia. EMBO Rep. 2, 239-243.
Pasparakis, M., Luedde, T. and Schmidt-Supprian, M. (2006). Dissection of the NFB signalling cascade in transgenic and knockout mice. Cell Death Differ. 13, 861-872.
Ryoo, H. D., Bergmann, A., Gonen, H., Ciechanover, A. and Steller, H. (2002).
Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat.
Cell Biol. 4, 432-438.
Sawa, Y., Arima, Y., Ogura, H., Kitabayashi, C., Jiang, J. J., Fukushima, T.,
Kamimura, D., Hirano, T. and Murakami, M. (2009). Hepatic interleukin-7 expression
regulates T cell responses. Immunity 30, 447-457.
Schmidt-Ullrich, R., Aebischer, T., Hulsken, J., Birchmeier, W., Klemm, U. and
Scheidereit, C. (2001). Requirement of NF-B/Rel for the development of hair follicles
and other epidermal appendices. Development 128, 3843-3853.
Sokol, N. S. and Cooley, L. (1999). Drosophila filamin encoded by the cheerio locus is
a component of ovarian ring canals. Curr. Biol. 9, 1221-1230.
Steller, H. (2008). Regulation of apoptosis in Drosophila. Cell Death Differ. 15, 11321138.
Stramer, B., Wood, W., Galko, M. J., Redd, M. J., Jacinto, A., Parkhurst, S. M.
and Martin, P. (2005). Live imaging of wound inflammation in Drosophila embryos
reveals key roles for small GTPases during in vivo cell migration. J. Cell Biol. 168,
567-573.
Sugiura, K., Taketani, S., Yoshimura, T., Nishino, T., Nishino, N., Fujisawa, J., Hisha,
H., Inaba, T. and Ikehara, S. (2007). Effect of hepatocyte growth factor on long term
hematopoiesis of human progenitor cells in transgenic-severe combined
immunodeficiency mice. Cytokine 37, 218-226.
Tzou, P., Ohresser, S., Ferrandon, D., Capovilla, M., Reichhart, J. M., Lemaitre, B.,
Hoffmann, J. A. and Imler, J. L. (2000). Tissue-specific inducible expression of
antimicrobial peptide genes in Drosophila surface epithelia. Immunity 13, 737-748.
Tzou, P., Reichhart, J. M. and Lemaitre, B. (2002). Constitutive expression of a single
antimicrobial peptide can restore wild-type resistance to infection in immunodeficient
Drosophila mutants. Proc. Natl. Acad. Sci. USA 99, 2152-2157.
van Hogerlinden, M., Rozell, B. L., Toftgard, R. and Sundberg, J. P. (2004).
Characterization of the progressive skin disease and inflammatory cell infiltrate in mice
with inhibited NF-B signaling. J. Invest. Dermatol. 123, 101-108.
Whalen, A. M. and Steward, R. (1993). Dissociation of the dorsal-cactus complex and
phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal.
J. Cell Biol. 123, 523-534.
Yu, C. Z., Hisha, H., Li, Y., Lian, Z., Nishino, T., Toki, J., Adachi, Y., Inaba, M., Fan,
T. X., Jin, T. et al. (1998). Stimulatory effects of hepatocyte growth factor on hemopoiesis
of SCF/c-kit system-deficient mice. Stem Cells 16, 66-77.
Zettervall, C. J., Anderl, I., Williams, M. J., Palmer, R., Kurucz, E., Ando, I. and
Hultmark, D. (2004). A directed screen for genes involved in Drosophila blood cell
activation. Proc. Natl. Acad. Sci. USA 101, 14192-14197.