Commentary 1373
Introduction
Ever since Boman et al. (Boman et al., 1972) published their
seminal paper showing that Drosophila melanogaster produced
antibacterial agents in reaction to infection, research into the insect
immune response has led to significant breakthroughs and
underscored Drosophila as a suitable model system for studying
the evolution of innate immunity. When Drosophila are invaded by
pathogenic organisms, such as bacteria or fungi, induction of the
immune response leads to the secretion of antimicrobial peptides
into the haemolymph, and circulating immunosurveillance cells
(haemocytes) attempt to phagocytise the invaders (Fig. 1). Parasites
too large to undergo phagocytosis, such as eggs laid by
endoparasitic wasps, provoke an encapsulation response, which
involves the adhesion of numerous haemocytes around the invader,
as well as inducing a melanisation response. Research has also
established Drosophila as a valuable model for studying the innate
immune response against viral pathogens (Kemp and Imler, 2009),
although the contribution of circulating immune cells against
viruses emerged only recently (Costa et al., 2009).
Although the production of antimicrobial peptides, and other
host defence factors, mainly relies on fat body cell function, cellular
immunity is provided by the haemocyte lineage, comprising three
broad subtypes of cells – the plasmatocytes, crystal cells and
lamellocytes – with each providing specific functions, namely
phagocytosis, coagulation and encapsulation, respectively (Fig. 2).
Haematopoiesis begins in the embryonic head mesoderm and gives
rise to two haemocyte cell lineages: the plasmatocytes and crystal
cells (Fossett et al., 2001; Fossett et al., 2003; Lebestky et al.,
2000; Milchanowski et al., 2004; Waltzer et al., 2003). Embryonic
plasmatocytes are involved in the phagocytosis of apoptotic bodies
and bacteria, and in wound healing (Moreira et al., 2010; Stramer
et al., 2005; Tepass et al., 1994; Vlisidou et al., 2009; Wood et al.,
2006). They also produce antimicrobial peptides and secrete
components of the extracellular matrix (ECM) (Lemaitre and
Hoffmann, 2007; Martinek et al., 2008). In larvae, haemocytes are
located in three main compartments: first in circulation; second in
a haematopoietic organ, the lymph gland, which consists of multiple
pairs of lobes and is located behind the brain; and third as a sessile
haemocyte population found just underneath the larval cuticle
(Crozatier and Meister, 2007; Lemaitre and Hoffmann, 2007;
Williams, 2007). In healthy larvae, plasmatocytes are the most
abundant haemocytes in circulation and are involved in
phagocytosis, encapsulation and the production of antimicrobial
peptides (Crozatier and Meister, 2007; Lemaitre and Hoffmann,
2007; Williams, 2007). Crystal cells make up the remaining
circulating haemocytes and, owing to their ability to rupture and
release components of the phenol oxidase cascade, are indispensable
for the melanisation of invading organisms, for wound repair
and for coagulation (Bidla et al., 2007; Meister, 2004). Melanisation
involves a complex series of reactions that converts tyrosine into
melanin, through phenol oxidase and other enzymes (Christensen
et al., 2005). In addition to its role in coagulation and wound
repair, insects also use melanisation as a means to confine parasites
inside a hardened proteinaceous capsule. The third type of
haemocyte, lamellocytes, are rarely seen in healthy larvae, but they
circulate in large numbers after parasitisation. Lamellocytes are
Summary
Research during the past 15 years has led to significant breakthroughs, providing evidence of a high degree of similarity between insect
and mammalian innate immune responses, both humoural and cellular, and highlighting Drosophila melanogaster as a model system
for studying the evolution of innate immunity. In a manner similar to cells of the mammalian monocyte and macrophage lineage,
Drosophila immunosurveillance cells (haemocytes) have a number of roles. For example, they respond to wound signals, are involved
in wound healing and contribute to the coagulation response. Moreover, they participate in the phagocytosis and encapsulation of
invading pathogens, are involved in the removal of apoptotic bodies and produce components of the extracellular matrix. There are
several reasons for using the Drosophila cellular immune response as a model to understand cell signalling during adhesion and
migration in vivo: many genes involved in the regulation of Drosophila haematopoiesis and cellular immunity have been maintained
across taxonomic groups ranging from flies to humans, many aspects of Drosophila and mammalian innate immunity seem to be
conserved, and Drosophila is a simplified and well-studied genetic model system. In the present Commentary, we will discuss what
is known about cellular adhesion and migration in the Drosophila cellular immune response, during both embryonic and larval
development, and where possible compare it with related mechanisms in vertebrates.
Key words: Cellular immunity, Haemocyte, Integrin, Phagocytosis, Parasitisation, Small GTPase
Journal of Cell Science 124, 1373-1382
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.064592
Drosophila cellular immunity: a story of migration and
adhesion
Marie-Odile Fauvarque1,2,3,
* and Michael J. Williams4,5,
*
1
CEA, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biologie à Grande Echelle, F-38054 Grenoble, France
2
INSERM, U1038, F-38054 Grenoble, France
3
UJF-Grenoble 1, F-38041 Grenoble, France
4
Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK
5
Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
*Authors for correspondence (marie-odile.fauvarque@cea.fr; m.j.williams@abdn.ac.uk)
JournalofCellScience
larger than other haemocytes and seem to be a specialised cell type
that is involved in the encapsulation of foreign pathogens that are
too large to undergo phagocytosis (Meister, 2004; Rizki and Rizki,
1992; Williams, 2007). Recently, it was demonstrated that, in
addition to their genesis in the larval lymph gland, many
lamellocytes derive directly from plasmatocytes (Fig. 2) (Honti
et al., 2010; Stofanko et al., 2010).
To date, most of our knowledge on phagocytosis and cell
migration in response to infection or tissue damage comes from
studies in human cell culture (Groves et al., 2008; Dupuy and
Caron, 2008). Chemotaxis and phagocytosis have also been
extensively studied in the unicellular free-living amoeba
Dictyostelium discoideum, which actively feeds on bacteria by
phagocytosis, thus enabling the deciphering of crucial mechanisms
and molecules involved in cell chemotaxis and bacterial
phagocytosis and killing (Cosson and Soldati, 2008; Jin et al.,
2009; Bozzaro et al., 2008; Lee et al., 2010). Complementary
studies show that Drosophila is also a particularly relevant model
organism for genetic in vivo studies of phagocytic cell function
during development and for studies on the elimination of pathogens
or transformed cells (Stuart and Ezekowitz, 2008). One of the
main advantages of using Drosophila for studying cell immune
functions, compared with using other invertebrate models, is the
complexity of its immune response. Indeed, Drosophila immunity
relies on interconnected humoural and cellular processes, which
both show striking similarities with those in mammalian innate
immunity (Lemaitre and Hoffmann, 2007; Ferrandon et al., 2007).
Studies using the embryonic Drosophila cellular immune system
confirmed that it was a relevant model system for understanding
the activity of circulating immunosurveillance cells during
developmental processes, wound healing and the host response to
infection. Moreover, specific advantages of using Drosophila as a
model system for the cellular immune response are the abilities to
follow cell migration in vivo (Stramer et al., 2005) and to assess
the contribution of immune cells in the defence against infection
(Stramer et al., 2005; Tingval et al., 2001). In this Commentary,
we discuss the current knowledge regarding the Drosophila
embryonic and larval cellular immune response in the context of
cellular adhesion and migration, and where possible compare it
with related mechanisms in vertebrates.
Migration of Drosophila embryonic
macrophages
Real-time studies in living Drosophila embryos have demonstrated
that the migration of differentiated plasmatocytes depends on
similar growth factors to that of mammalian blood phagocytic
cells (Stramer et al., 2005). Notably, developmentally controlled
plasmatocyte migration requires the Drosophila platelet-derived
growth factor (PDGF) and vascular endothelial growth factor
(VEGF) receptor Pvr, which, in mammals, directs neutrophils
and/or macrophage migration during development and in response
to infection (Brückner et al., 2004; Cho et al., 2002; Duchek et al.,
2001; Heino et al., 2001). Pvr has three potential ligands, the
PDGF- and VEGF-related factors 1 to 3 (Pvf1, Pvf2 and Pvf3); of
these, Pvf2 and Pvf3 contribute to embryonic plasmatocyte
migration (Brückner et al., 2004; Munier et al., 2002; Olofsson and
Page, 2005; Wood et al., 2006). Developmental migration of
embryonic plasmatocytes occurs in three distinct stages. First,
plasmatocytes migrate out of the cephalic (head) mesoderm to
populate the head region of the embryo. In the next phase,
plasmatocytes leave the head region and follow Pvf-regulated
routes around the embryo, including along the ventral nerve chord
(VNC) and the embryonic dorsal vessel (heart) (Fig. 3). During
this process, plasmatocytes, in a manner similar to mammalian
macrophages, start to ingest apoptotic bodies that arise from
naturally occurring developmental processes (Tepass et al., 1994).
In the latter stages of embryogenesis, plasmatocytes are found
scattered throughout the embryo, but maintain their ability to
migrate to wound sites (Fig. 3) (Moreira et al., 2010; Paladi and
Tepass, 2004; Tepass et al., 1994; Wood et al., 2006).
The molecular basis of plasmatocyte migration during
development has been elucidated by in vivo genetic studies. It has
1374 Journal of Cell Science 124 (9)
Plasmatocyte
Crystal
cellLamellocyte
Fat
body
Wasp egg Fungi Bacteria Viruses
Cellular response Humoural response
Antimicrobial peptide secretion
Phagocytosis
Encapsulation
Coagulation
Antimicrobial peptide secretion
Haemocytes
Fig. 1. Schematic representation of the Drosophila larval immune
response. Microbial infections initiate responses by both the cellular and
humoural immune tissues. Haemocytes and the fat body can produce and
secrete antimicrobial peptides in response to bacterial and fungal infections.
Both haemocytes and the fat body might be involved in the anti-viral response,
whereas haemocytes are essential for the anti-parasitic encapsulation response.
Plasmatocyte Lamellocyte
Antimicrobial peptide
secretion
Phagocytosis
Encapsulation
Encapsulation
Crystal cell
Encapsulation
Coagulation
Fig. 2. Drosophila haemocyte subtypes. Plasmatocytes resemble the
mammalian monocyte macrophage lineage and are involved in phagocytosis,
encapsulation and the production of antimicrobial peptides. Lamellocytes,
which are rarely seen in healthy larvae, are larger than other haemocytes and
are involved in the encapsulation of invading pathogens. Many lamellocytes
derive directly from plasmatocytes, as indicated by the arrow. Crystal cells
rupture to release components of the phenol oxidase cascade, involved in the
encapsulation process of invading organisms, coagulation and wound repair.
The image of the crystal cell has been kindly provided by Ulrich Theopold.
JournalofCellScience
been proposed that Pvf3 is the first Pvr ligand that is involved, and
that it regulates the migration of plasmatocytes out of the head
region. Indeed, Pvf3 is expressed in the VNC earlier in development
than Pvf2, and in Pvf3 mutant flies plasmatocytes do not begin
their migration along the VNC (Table 1) (Wood et al., 2006).
Although the signalling pathways that drive plasmatocyte migration
downstream of Pvr are still not fully established, the small GTPases
Rac1 and Rac2 are good candidates for mediating Pvr-dependent
signalling; in Drosophila embryos lacking the activity of these two
GTPases, plasmatocytes fail to migrate along the posterior end of
the VNC (Fig. 3). Furthermore, Rac1 is required downstream
of Pvr for border cell migration during Drosophila oogenesis and
for thorax closure during metamorphosis; thus, it is likely to play
a similar role during plasmatocyte migration (Duchek et al., 2001;
Ishimaru et al., 2004; Mathieu et al., 2007). Interestingly, a similar
pathway (i.e. PDGF receptor signalling through the small GTPase
Rac1) controls directional cell migration in mammalian cells
(Kawada et al., 2009). In mammalian NIH3T3 cells, the PDGF
receptor sequentially activates Ras-related protein 1 (Rap1) and
Rac1 to induce lamellipodia formation, and this activation is
necessary for PGDF-regulated cell migration in the cell line
(Takahashi et al., 2008). Similarly, Drosophila Rap1 and its
activator PDZ-GEF, also known as Dizzy, are necessary for the
migration of embryonic haemocytes along the posterior half of
the VNC (Huelsmann et al., 2006; Paladi and Tepass, 2004; Wood
et al., 2006). These results suggest that Pvr signals through Rap1,
Rac1 and Rac2 to coordinate the second stage of plasmatocyte
migration in response to activation by the Pvf2 ligand (Fig. 3).
However, the initial Pvf3-induced migration of plasmatocytes
out of the head region does not require the Rac1 and Rac2 GTPases.
This indicates that other factors downstream of Pvr might control
plasmatocyte migration in response to Pvf3 during the initial stages
of migration out of the head region. In mammalian cells, receptor
tyrosine kinases, including the PDGF receptor, signal through Ras
to regulate cell proliferation, cell polarity and cell migration
(Lahsnig et al., 2009; Ogita et al., 2009). By analogy, in Drosophila
embryos, Pvr signalling in response to Pvf3 expression during the
initial phases of plasmatocyte migration might occur through
the Drosophila Ras protein Ras85D. Indeed, a deletion removing
two closely located GTPase-encoding genes, Ras85D and the
insect-specific Rho-family GTPase RhoL, prevents plasmatocytes
from migrating out of the head region (Paladi and Tepass, 2004).
As a mutation in Ras85D is known to affect larval haemocyte
behaviour and cell morphology, Ras85D might be required for
1375Drosophila cellular immunity
Pvf2,Pvr; RhoL
Pvf2,Pvr; Rac1 and Rac2?
PI3K; Rac1 and Rac2,Rho1,Cdc42
Pvf3,Pvf2,Pvr; Ras85D
H2O2
H2O2
H2O2
Ventral nerve cord
Wound
site
Anterior Posterior
Dorsal
Ventral
Fig. 3. Schematic illustration of the pathways controlling embryonic plasmatocyte migration during normal development and after wounding. The
illustration shows a Drosophila embryo at late embryogenesis. Pvf3 and Pvr might signal through the small GTPase Ras85D to initiate plasmatocyte (represented
by the blue circles) migration along the anterior portion of the VNC. Later in development, Pvf2 signals through Pvr and possibly also through Rac1 and Rac2
(indicated by a question mark) and results in plasmatocyte migration along the posterior VNC (along the orange arrow). The wound response is illustrated in the
upper right-hand corner of the embryo (light blue). After epithelial wounding, the damaged cells or cells near the wound (red area) release H2O2, which is sensed by
plasmatocytes and initiates their migration to the wound site (along the blue arrow). Migration to wound sites does not require Pvr signalling, but instead relies on
PI3K activity and the small GTPases.
Table 1. Drosophila developmental stages and plasmatocyte migration
Stage of
development
Hours after
fertilisation Developmental process Plasmatocyte migration
Stage 10 4.0–5.0 Gnathal and clypeolabral lobe formation (head features) Plasmatocytes can be first identified
Stage 11 5.0–7.0 Epidermal parasegmentation evident; mesectodermal cell
ingress; end of third postblastoderm mitosis; end of
neuroblast formation
Plasmatocytes migrate throughout the head region; Pvf3
expressed in VNC
Stage 12 7.0–9.5 Germ band retraction; ventral closure; segment formation;
fusion of anterior and posterior midgut
Plasmatocytes start spreading throughout the embryo; Pvf2
expressed in anterior portion of the VNC; Pvf-induced
migration along the anterior portion of the VNC
Stage 13 9.5–10.0 End of germ band retraction; central nervous system and
peripheral nervous system differentiation
Pvf2 expressed along entire VNC; plasmatocytes migrate along
VNC
Stage 14 10.0–11.0 Dorsal closure of epidermis; head involution begins Beginning anteriorly and moving in a posterior direction; Pvf2
RNA levels decrease in the VNC; plasmatocytes migrate
along the posterior portion of VNC
Stage 15 11.0–13.0 End of dorsal closure; head involution; cuticle deposition
begins
Plasmatocytes are evenly distributed throughout the embryo
Hatching 21–22 Hatching to first-instar larva
JournalofCellScience
embryonic haemocyte migration (Bakal et al., 2007; Rogers et al.,
2003; Zettervall et al., 2004). It has been demonstrated that RhoL
is not necessary for Pvr-induced plasmatocyte migration along the
VNC (Paladi and Tepass, 2004; Siekhaus et al., 2010).
Taken together, however, the above results suggest that
Drosophila Pvr activates Ras and Rac GTPases (Rap1, Ras85D,
Rac1 and Rac2) to ensure appropriate cellular migration in response
to developmental signals, in a manner similar to that with the
mammalian PDGF receptor.
Among the underlying processes that are absolutely required for
cell migration and transmigration is integrin-mediated cell adhesion
at the leading edge of the cell (Caswell et al., 2009). A recent study
demonstrated that, in Drosophila embryonic plasmatocytes, RhoL
interferes with Rap1 GTPase-induced integrin adhesion, by inhibiting
the localisation of Rap1 to the leading edge. Inhibition of integrinbased
adhesion is necessary to regulate the cadherin interactions that
allow plasmatocytes to transmigrate from the head region, through
the epithelium, to the posterior of the embryo (Siekhaus et al., 2010).
The molecular events underlying this transmigration are very similar
to those in the migration of vertebrate immune cells during
inflammation (Basoni et al., 2005; Ebisuno et al., 2009; M’Rabet
et al., 1998). In fact, although developmental and pro-inflammatory
cytokines use different types of receptors in Drosophila and
mammalian phagocytic cells, they employ common downstream
effectors for driving cell adhesion changes during migration.
Migration of embryonic macrophages in response to
epithelial wounding
In mammals, studies have shown that leukocyte polarisation and
migration in response to wounding can be induced by a variety
of factors, including cytokines, ATP, bacterial factors (e.g.
lipopolysaccharides and peptidoglycans) and ECM breakdown
products (Hammer, 2005; Jones, 2000). Many of these factors
stimulate G-protein-coupled receptor (GPCR) pathways and lead
to the activation of phosphoinositide 3-kinase (PI3K). One major
role of PI3K is to phosphorylate phosphatidylinositol (4,5)bisphosphate
[PtdIns(4,5)P2] to give rise to phosphatidylinositol
(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (Cantley, 2002).
PtdIns(3,4,5)P3 is known to serve as a docking site for the Dblhomology
domain and Dock-type Rho guanine-nucleotideexchange
factors (RhoGEFs), as well as for the serine/threonine
protein kinase Akt (also known as PKB). The recruitment of
RhoGEFs leads to localised activation of Rho GTPase pathways
that are involved in inducing protrusions, such as filopodia and
lamellopodia, and thus in directional movement (Alahari, 2003;
Kolsch et al., 2008; Lavenburg et al., 2003; Sachdev et al., 2002).
A number of elegant studies, in which the epithelium of
developing Drosophila embryos was wounded with a laser, have
assessed the ability of plasmatocytes to migrate to the wound site
in a living organism (Stramer et al., 2005; Wood et al., 2006).
Upon tissue damage, plasmatocytes migrate to the wound site in a
manner that is independent of the Pvf–Pvr pathway but is dependent
on PI3K, demonstrating that plasmatocytes are able to distinguish
between developmental cytokines, such as Pvf2 and Pvf3, and
PI3K-mediated wound-induced signals (Wood et al., 2006). The
receptor that activates PI3K in embryonic plasmatocytes is yet to
be identified, but it is possible that, similar to the pathway in
mammalian leukocytes, Drosophila plasmatocytes also use a
GPCR-coupled pathway to recognise as yet unknown woundinduced
signals, which redirect them from their developmentally
regulated Pvf–Pvr-mediated pathway of migration towards the site
of wounding. Wood and collaborators have also attempted to
determine whether additional chemotactic signals, known to attract
macrophages to wound sites, elicit a haemocyte response to
wounding [e.g. ATP, epidermal growth factor (EGF) and fibroblast
growth factor (FGF)], but found that only H2O2 was able to redirect
plasmatocytes to the wound site (Moreira et al., 2010). It should
be noted that, similar to mammalian embryos (Morris et al., 1991;
Wood et al., 2000), there is a refractile period early in haemocyte
development, in which haemocytes do not respond to H2O2, and
that they only become responsive to wound signalling after stage
15 (mid-to-late embryogenesis) (Moreira et al., 2010).
In the future, the knowledge gained from the above-mentioned
initial studies will allow researchers to, for instance, use the
Drosophila embryonic cellular immune system to understand how
circulating immunosurveillance cells differentiate between, and
prioritise, two competing signals in physiologically relevant
situations in vivo.
Cytoskeleton regulatory proteins control larval
haemocyte cell shape changes
During the course of an infection, immune cells migrate towards
the site of microbial entry with the aim of eliminating pathogens
and, furthermore, contributing to the repair of the wound caused
by the microbe (Martin and Leibovich, 2005; Nishio et al., 2008).
This process requires not only cell migration but also changes in
cell adhesion, as well as phagocytosis, which universally depend
on the dynamics of the actin network (i.e. the polymerisation or
depolymerisation of actin filaments) (Fig. 4). Briefly, actin
dynamics mainly relies on the Arp2/3 complex, a few nucleationpromoting
factors and formins, which control filament
polymerisation and depolymerisation through interactions with
regulatory proteins (Campellone and Welch, 2010). Actin nucleation
depends upon the activation of Arp2/3, which directly binds to
members of the Wiskott–Aldrich syndrome protein (Wasp) family,
including Scar, whereas debranching and depolymerisation of actin
filaments are controlled by cofilin and cofilin-like proteins (Chan
et al., 2009). The concerted activity of Rho-family GTPases (e.g.
Rho, Rac and Cdc42) then directs the formation of different cellular
protrusions, such as filopodia, which contain narrow actin
projections (so-called actin spikes), membranes ruffles or large
lamellipodial extensions (Etienne-Manneville and Hall, 2001;
Nobes and Hall, 1995). As might be expected, studies in cultured
Drosophila S2 cells (a cell line derived from embryonic
haemocytes) or primary larval plasmatocytes revealed a similar
requirement for cytoskeleton regulatory proteins to that previously
found in mammalian cells during the processes of migration (see
above), phagocytosis and the control of cell shape changes upon
their spreading on glass (Table 2). A reverse genetic approach that
investigated the contribution of 90 cytoskeleton regulatory proteins
in lamellopodia formation in S2 cells, through systematic RNA
interference (RNAi), has demonstrated the requirement for actin
nucleation proteins (Arp2/3 and Scar), capping proteins, filament
depolymerisation factors [cofilin and actin-interacting protein 1
(Aip1)] and actin-monomer-binding proteins (profilin and cyclaseassociated
protein) (Rogers et al., 2003). Moreover, initiation of
cell spreading requires Rac GTPases and the adaptor protein
Dreadlocks (Dock), which is a known stimulator of Scar and
Arp2/3 (Kunda et al., 2003; Rogers et al., 2003). Direct genetic
approaches have also revealed the necessity of cytoskeleton
regulatory proteins in mediating phagocytosis. For example, the
Drosophila Scar mutant was isolated in a screen that focused on
1376 Journal of Cell Science 124 (9)
JournalofCellScience
the phagocytic properties of circulating primary macrophages
isolated from mutant larvae. Drosophila Scar, and the closely
related protein Drosophila Wasp, is required for the internalisation
of Escherichia coli and Staphylococcus aureus particles (Pearson
et al., 2003; Rogers et al., 2003). A second mutation, in chickadee
(chic), was also isolated in the screen; the chic mutant macrophages
exhibit an opposite phenotype, leading to enhanced engulfment
properties (Rogers et al., 2003). The chic gene encodes the
Drosophila homologue of profilin, and mammalian profilin was
shown to bind and regulate Scar in vitro (Pollard and Borisy, 2003)
and to promote actin dynamics in vivo (Bottcher et al., 2009;
Witke, 2004). In addition to causing alterations in phagocytosis,
the circulating plasmatocytes of both scar and chic mutant larvae
display aberrant actin cytoskeleton structures and abnormal shape.
Specifically, the plasmatocytes of scar mutants are enlarged and
exhibit numerous actin spikes, whereas chic mutant cells spread
more widely on glass, when compared with spreading of wild-type
cells, and can be distinguished by a large lamellipodium around
the cell. Therefore, it has been speculated that abnormal cell shape
and defects in actin dynamics could be the underlying reason for
the altered cell adhesion and phagocytosis observed in mutant
plasmatocytes.
Although studies in Drosophila have permitted in vivo
investigation of the function of cytoskeleton regulatory proteins,
purposeful studies to decipher their activity in the physiological
context, such as in the immune response, had largely not been
undertaken. Along these lines, our groups and others have
performed detailed analyses of the in vivo function of the Rhofamily
GTPases in haemocytes in order to show that they make
essential and non-redundant contributions to embryonic migration
(see above), to the cell shape changes of haemocytes and to
bacterial phagocytosis. Notably, expression of wild-type and mutant
forms of Rac1 in Drosophila specifically in plasmatocytes revealed
that Rac1 induces F-actin accumulation and lamellipodia formation
through two distinct pathways involving either the Jun kinase
Basket (Bsk) or Twinstar (the Drosophila cofilin homologue)
(Williams et al., 2006). A complementary study has shown that
Rac1 induces Rho1 activation and F-actin stress fibre formation,
which allows filopodia to differentiate, and, moreover, that Rac2
is also required for this process (Williams et al., 2007). Finally,
experiments with injected or in-vivo-expressed bacterial toxins
that target GTPases also confirm that Rho GTPases contribute to
embryonic haemocyte motility (Vlisidou et al., 2009) and to larval
and adult haemocyte-dependent phagocytosis of bacteria (AvetRochex
et al., 2005; Avet-Rochex et al., 2007). Such in vivo
models, in which a bacterial toxin is either injected into the living
Drosophila embryos or expressed in host cells by transgenic means,
open new avenues to study the functions of bacterial toxins in
vivo. These experiments might also lead to the discovery of new
host genes and proteins whose functions are modified by pathogens
and which are therefore likely to be involved in innate immunity.
Although most reverse genetic approaches were initiated from
data obtained from human cell culture models, a further protein
involved in cell adhesion and phagocytosis in Drosophila was
deduced using data obtained in the phagocytic unicellular organism
D. discoideum. The nonaspanin TM9SF4, also known as Phg1A,
is an evolutionarily conserved protein of nine transmembrane
segments, whose function in adhesion and phagocytosis was first
described in Dictyostelium (Benghezal et al., 2003; Cornillon et al.,
2000) and subsequently also observed in Drosophila haemocytes
1377Drosophila cellular immunity
Rac1,Rac2
Arp2/3
Scar
Phagocytic
receptors and
co-receptors
F-actin
αβ αβ αβ
IntegrinsTM9SF4
Rac2
Adhesion
Wasp egg encapsulation Phagocytosis
Wasp egg
αβ
A B
Nrg
Plasma
membrane
Integrins
TM9SF4
NimC1 PGRP-LC
Bacteria
Phagocytic
cup
Fig. 4. Pathways inducing changes in haemocyte shape upon infection. (A)Integrin PS defective (mys mutant) (Irving et al., 2005), TM9SF4 (Bergeret et al.,
2008) and Rac2 (Williams et al., 2005) mutant larvae fail to encapsulate wasp eggs properly following larval parasitisation, and their larval plasmatocytes display
either defective adhesion and/or phagocytosis defects associated with an abnormal F-actin cytoskeleton network. This suggests that Rac2 plays an essential
function in signal transduction from adhesion transmembrane proteins to the cytoskeleton during the encapsulation processes. Similar to platelets during
thrombosis, plasmatocytes require the L1CAM homologue Neuroglian (Nrg) to properly adhere to and spread over parasitoid wasp eggs (Williams, 2009).
(B)Phagocytosis of smaller pathogens (such as bacteria or yeast, shown in red) depends upon their opsonisation by circulating receptors or complement-like
proteins (not shown) (Garver et al., 2006; Strochein-Stevensen et al., 2006), and on their recognition by transmembrane phagocytic receptors, such as PGRP-LC,
Eater or NimC1 (Ramet et al., 2002; Kocks et al., 2005; Kurucz et al., 2007). How exactly phagocytic receptors induce the formation of a phagocytic cup and actin
reorganisation required for pathogen engulfment has been poorly investigated to date in Drosophila. Putatively, phagocytic receptors might interact with coreceptors,
such as integrins, which are also required for phagocytosis, thereby possibly transmitting an internalisation signal to Rho GTPases that regulate the
cytoskeleton. Deciphering the signalling molecules between these receptors and the cytoskeleton regulatory proteins known to be required for phagocytosis, such
as the Rac1 and Rac2 (dashed arrow), is one of the future challenges to better understand Drosophila cellular immunity. Rho GTPases, notably Rac1 and Rac2,
affect haemocyte cell shape changes, upon infection, by activating cytoskeleton-associated proteins, such as Scar and Arp2/3.
JournalofCellScience
(Bergeret et al., 2008) and in human tumour cells (Lozupone et al.,
2009). Similar to the phenotype of scar and Rac1 mutants,
Drosophila TM9SF4 mutant haemocytes are larger than control
haemocytes and can be distinguished by their numerous actin
spikes. Drosophila TM9SF4 mutant larvae also fail to correctly
encapsulate parasitoid wasp eggs (Bergeret et al., 2008), a process
that requires Rac2 and integrin-mediated adhesion of plasmatocytes
to the foreign parasite (Irving et al., 2005; Williams et al., 2005).
Owing to the phenotypic similarities to those induced by mutant
components of the cytoskeleton regulatory networks, such as
Myospheroid (a Drosophila integrin ), Rac1, Rac2, Scar and
Twinstar (Drosophila cofilin), TM9SF4 is considered a candidate
for coupling changes in the actin cytoskeleton to adhesion and,
putatively, for controlling integrin-dependent activation of Rho
1378 Journal of Cell Science 124 (9)
Table 2. Comparison of a non-exhaustive set of proteins regulating cell shape in Drosophila haemocytes and their mammalian
counterparts
Proteins [mammalian
name (Drosophila
homologue)]
Function in Drosophila
haemocytes References
Known functions in mammalian
phagocytes References
Membrane proteins
-integrin
(Myospheroid)
Adhesion; encapsulation (Irving et al., 2005) Adhesion; migration; signalling (reviewed by Dupuy and
Caron, 2008)
L1CAM (Neuroglian) Adhesion (Nardi et al., 2006) Cell adhesion; homophilic and
heterophilic interactions
(reviewed by Hortsch, 2000)
Encapsulation (Williams, 2009) Cell migration (Maddaluno et al., 2009)
Platelet aggregation (Prevost et al., 2002)
TM9SF4 (TM9SF4 or
Phg1A)
Adhesion; phagocytosis;
encapsulation
(Bergeret et al., 2008) Phagocytosis and cannibalism (in
tumour cells)
(Lozupone et al., 2009)
Rho GTPases
Common functions Haemocyte migration (Paladi and Tepass, 2004) Adhesion; migration;
phagocytosis; cytoskeleton
dynamics; integrin complex
assembly; FAK turnover
(reviewed by Ridley, 2001;
Bokoch, 2005)
Wound-induced migration (Kawada et al., 2009; Moreira
et al., 2010; Wood et al.,
2006)
Macrophage adhesion and
migration
(Ridley, 2007)
Adhesion (Stramer et al., 2005) Neutrophil chemotaxis (Zhang et al., 2009)
Phagocytosis (Avet-Rochex et al., 2007)
Rac1 Recruitment of sessile
haemocytes upon
immune challenge
(Williams et al., 2005;
Williams et al., 2006)
Cell spreading; membrane
ruffling; phagocytosis
(Wells et al., 2004; Cox et al.,
1997)
Filopodia and lamellipodia
differentiation
Platelet aggregation (McCarty et al., 2005; Ridley,
2007)
Rac2a
Phagocytosis and host
defence
(Avet-Rochex et al., 2007) NADPH oxidase activation (Knaus et al., 1991)
Encapsulation (Williams et al., 2005;
Williams et al., 2006)
Podosome formation (Linder and Aepfelbacher,
2003; Ridley, 2007)
Cdc42 Cell polarity; velocity (Stramer et al., 2005) Cell chemotaxis (Allen et al., 1998)
Cell shape (Rogers et al., 2003) Cell polarity (Etienne-Manneville and Hall,
2001)
Wasp activation (Park and Cox, 2009)
Rho1 Tail retraction (Stramer et al., 2005) Cell migration; tail retraction (Worthylake et al., 2001)
Dynamics of cell–cell
contact
Other cytoskeleton
regulatory molecules
SCAR (SCAR) Phagocytosis; lamellipodia (Pearson et al., 2003; Rogers
et al., 2003)
Actin nucleation; activation of
Arp2/3 complex
(Machesky et al., 1999)
Arp2/3 Phagocytosis; lamellipodia (Pearson et al., 2003; Rogers
et al., 2003)
Actin nucleation (Etienne-Manneville and Hall,
2002)
Polarisation and migration (Linder et al., 2000)
Activation of Rho GTPases (reviewed by Campellone and
Welch, 2010)
Cofilin (Twinstar) Lamellipodia; spreading (Rogers et al., 2003) Arp2/3 dissociation; actin
branching
(Chan et al., 2009)
Profilin (Chickadee) Lamellipodia; spreading;
phagocytosis
(Pearson et al., 2003; Rogers
et al., 2003)
Actin monomer binding; actin
assembly
(Coppolino et al., 2001;
Machesky et al., 1999)
Diaphanous Filopodia; Rho-dependent
signalling
(Williams et al., 2007) Actin polymerisation; phagocytic
cup; Rho-dependent signalling
(Colucci-Guyon et al., 2005;
Brandt et al., 2007)
Signalling molecules
Rho kinase Rac1 activation; actin
stress fibres
(Williams et al., 2007) Rho-induced actin reorganisation (Watanabe et al., 1999)
Slingshot Lamellipodia formation; Factin
distribution
(Rogers et al., 2003) Dephosphorylation of cofilin (Niwa et al., 2002)
a
Drosophila Rac2, also named Rac1b, is approximately equally related to human Rac1 [171 of 191 identical amino acids (89% identity)] and human Rac2
protein [169 of 192 identical amino acids (88% identity)].
JournalofCellScience
GTPases during the adhesion of plasmatocytes to pathogens (Irving
et al., 2005; Zhuang et al., 2008).
In addition, we note that there are several phagocytic receptors,
which have been discovered through genetic and reverse genetic
approaches, that mediate the recognition and internalisation of
various pathogens by larval plasmatocytes or Drosophila cultured
S2 cells. These include Eater, Nimrod C1 (NimC1), and the
peptidoglycan recognition proteins (PGRPs) PGRP-LC and PGRPSC1
(Kocks et al., 2005; Kurucz et al., 2007; Pearson et al., 2003;
Ramet et al., 2002; Garver et al., 2006). However, the signalling
pathways for the subsequent activation of Rho GTPases and
reorganisation of the actin network, which are required for the
initiation of the phagocytic cup, have not yet been elucidated in
Drosophila. Deciphering these pathways constitutes a challenge
for building an integrated view of pathogen recognition,
phagocytosis and killing in multicellular organisms.
The Drosophila encapsulation response has
aspects in common with thrombosis
The first step in mammalian thrombosis is the adhesion of platelets
to sites of endothelial damage in vessels where the ECM has been
exposed, and the activation of platelets by inflammatory triggers
might be a crucial component leading to atherothrombosis. Insertion
of a parasitoid wasp egg into the Drosophila larval open circulatory
system (haemocoel) mimics vascular injury as it results in
deposition of ECM onto the egg (Russo et al., 1996). Parasitisation
also induces changes in haemocyte morphology, and in their
inherent adhesive properties, thus allowing haemocytes to form a
cellular capsule around the ECM. In a manner similar to processes
occurring during thrombosis, cell–cell contacts between haemocytes
on the surface of the wasp egg promote the stabilisation of a
growing cellular capsule (Russo et al., 1996; Williams et al., 2005).
Analogous to activated platelets inducing the inflammation
response, which can enhance the development of the growing
thrombus, parasitisation of Drosophila larvae also gives rise to an
inflammatory state, which leads to the recruitment of additional
haemocytes into the circulation (Lanot et al., 2001; Markus et al.,
2009; Zettervall et al., 2004).
After attaching to the wasp egg, plasmatocytes change from a
rounded to a more spread morphology. In a process similar to
platelets during thrombosis, once plasmatocytes are attached and
start to spread on the wasp egg, they extend filopodia from their
cell periphery towards other plasmatocytes (Williams, 2009). After
spreading around the wasp egg, plasmatocytes form cellular
junctions, effectively separating the wasp egg from the larval
haemoceol (Russo et al., 1996; Williams et al., 2005). Next,
lamellocytes, the second class of circulating haemocytes, introduced
above, recognise the plasmatocytes surrounding the wasp egg.
Although it is evident that adhesion and change in cell shape are
an essential part of the cellular immune response against parasitoid
wasp eggs, we still do not fully understand the spatiotemporal
regulation of the signalling events that are involved in this response.
Towards this goal, Wertheim and colleagues performed a
microarray analysis of Drosophila genes that are upregulated after
parasitisation by the parasitoid wasp Asobara tabida (Wertheim
et al., 2005). The analysis revealed a number of genes that are
involved in cell adhesion and cytoskeleton regulation, including
genes encoding Hemolectin, a protein that contains a von
Willebrand factor domain and known to be involved in coagulation
(Lesch et al., 2007), two fibrinogen-like proteins, integrins and
tubulins, all of which are also centrally involved in mammalian
thrombosis (Jennings, 2009), further highlighting that both
processes are related. In addition, Irving and colleagues reported
that the Drosophila -integrin Myospheroid, and possibly the
-integrin PS4, is necessary for lamellocytes to correctly
encapsulate parasitoid wasp eggs (Irving et al., 2005). Furthermore,
our work has highlighted the importance of another adhesion
molecule, the Drosophila L1CAM homologue Neuroglian (Nrg),
in the encapsulation response (Williams, 2009). L1CAM was
previously shown to be necessary for platelet aggregation during
thrombus formation, where it interacts with integrins (Prevost et
al., 2002). The Rho-family GTPases Rac1 and Rac2 were shown
to be involved in the Drosophila cellular immune response against
the parasitoid wasp egg from Leptopilina boulardi (Williams et al.,
2005; Williams et al., 2006). Intriguingly, mammalian Rac1 is
known to be necessary for platelets to form a proper thrombus
(Akbar et al., 2007; McCarty et al., 2005). Gaining a thorough
understanding of the Drosophila anti-parasitoid response thus might
lead to it being used as an in vivo model to identify new antithrombotic
drugs.
Concluding remarks
Phagocytic cells must be capable of migration by chemotaxis, as
well as internalisation and digestion of external dead or live
material, regardless of whether they act as a unicellular organism,
such as free-living amoebae, or inside a multicellular organism.
The functions of phagocytic cells serve many diverse biological
processes, such as nutrition, tissue remodelling, pathogen
recognition and clearance, antigen presentation, cytokine secretion
and the elimination of altered cells or body parts from the organism
itself (Desjardins et al., 2005). This panel of functions relies on
protein-sensing complexes, adhesion molecules, signalling
pathways, membrane dynamics and cytoskeleton modifications
that are mostly evolutionarily conserved (Abedin and King, 2010;
Wang, 2009; Pollard and Cooper, 2009; Orlando and Guo, 2009;
Insall and Machesky, 2009). Although amoebae are extensively
used as the simplest genetic model to provide new insight into
molecular mechanisms of adhesion, chemotaxis and phagocytosis
(Abedin and King, 2010; Jin et al., 2009), the use of more complex
non-mammalian animals, such as nematodes (D’mello and Birge,
2010), Drosophila (Irving et al., 2005; Kocks et al., 2005; AvetRochex
et al., 2007; Williams et al., 2006; Stuart and Ezekowitz,
2008) and zebrafish (Herbomel et al., 1999; Lieschke et al., 2001;
Mathias et al., 2009), has provided novel tools for the investigation
of phagocyte function in developmental tissue remodelling, tissue
repair and host defence. Nematodes principally increased our
understanding of apoptotic cell clearance (Zhou and Yu, 2008;
Kinchen and Ravichandran, 2010), whereas Drosophila and
zebrafish additionally seem to be relevant models for the study of
innate immunity (in both cases) or adaptive immunity (in the case
of zebrafish). A main achievement of the past decade is undoubtedly
the development of live imaging in complex organisms, such as
nematodes, Drosophila (Stramer et al., 2005: Wood et al., 2006),
zebrafish (Levraud et al., 2009) and even mammalian models
(Coombes and Robey, 2010), for the study of macrophage
behaviour in various wild-type or mutant genetic contexts.
The field of Drosophila cellular immunity, in particular, has
witnessed significant developments over the past two decades,
expanding our general understanding of innate immunity. A number
of recent studies have demonstrated that the Drosophila cellular
immune response can help in our understanding of the mechanisms
involved in haematopoiesis, wound healing, thrombosis,
1379Drosophila cellular immunity
JournalofCellScience
immunosurveillance cell migration and immune activation. Results
obtained in Drosophila not only support data obtained in
mammalian cells, reinforcing our knowledge of immune cell
function, but have also helped us to gain knowledge regarding the
molecular mode of action of known proteins, as well as their roles
in vivo during development or in response to infection. Recently,
researchers have started to use the Drosophila cellular immune
response as a tool to define how innate circulating
immunosurveillance cells interact with tumours to restrict tumour
growth (Pastor-Pareja et al., 2008). These findings demonstrate
that, in the coming decades, Drosophila will continue to be a
powerful model for increasing our understanding of the
multifunctional innate cellular immune response.
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