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Review
Unraveling tissue repair immune responses in flies Brian M. Stramer a,∗ , Marc S. Dionne b a b
King’s College London, Randall Division of Cell and Molecular Biophysics, London SE1 1UL, United Kingdom King’s College London, Centre for the Molecular and Cellular Biology of Inflammation, London SE1 1UL, United Kingdom
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Keywords: Drosophila Wound repair
a b s t r a c t Drosophila melanogaster has emerged as a powerful model to understand innate immune responses to infection (note the 2011 Nobel Prize in Physiology or Medicine), and in recent years this system has begun to inform on the role and regulation of immune responses during tissue injury. Due to the speed and complexity of inflammation signals upon damage, a complete understanding of the immune responses during repair requires a combination of live imaging at high temporal resolution and genetic dissection, which is possible in a number of different injury models in the fly. Here we discuss the range of woundinduced immune responses that can be modeled in flies. These wound models have revealed the most immediate signals leading to immune cell activation, and highlighted a number of complex signaling cascades required for subsequent injury-associated inflammatory responses. What has emerged from this system are a host of both local acting signals, and surprisingly, more systemic tissue repair immune responses. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Most of what we know about immune responses during tissue damage has been gleaned from studies of human tissues and cells, or from mammalian models. However, the complexity of inflammation, and its dynamic nature make it problematic to dissect at a genetic level and difficult to live image. As a result, a number of basic model organisms are starting to be exploited. Ilya Metchnikov was the first to utilize a model organism, starfish larvae, to observe the inflammatory response to wounding [1]. While a simple organism like starfish, does not exhibit the Oxford English Dictionary’s definition of inflammation – rubor (redness), calor (heat), tumor (swelling), and dolor (pain) – the central etiology of its wound inflammation (which was highlighted in Ilya’s Nobel lecture) is the same: recruitment of professional microbe killing and debris clearing cells. Indeed, Drosophila, which is far more genetically tractable than Metchnikov’s starfish, has been used as a model of animal immunity for some time. Strikingly, key components of a Drosophila’s response to pathogenic infection, such as a requirement for Toll signaling, are evolutionarily conserved (and in 2011 a Nobel Prize in Physiology or Medicine was awarded for this discovery). While most of the Drosophila work has focused on
∗ Corresponding author. Tel.: +44 0207 848 6272. E-mail addresses: [email protected] (B.M. Stramer), [email protected] (M.S. Dionne).
infection responses, flies are increasingly being utilized to understand the role and regulation of immune responses during wound healing. This review will discuss the physiologically relevant questions that can be addressed in this model, and highlight recent discoveries that have exploited the benefits of this basic model system. 1.1. Drosophila immune cells Flies have a relatively simple immune system that is composed of hemocytes (insect “blood” cells) that can differentiate into just a few cell-types [2]. The major hemocyte population is the plasmatocyte, which is a macrophage-like phagocytic cell-type that is capable of migratory responses to tissue damage. In contrast, Crystal cells make up a small percentage of the total hemocyte population at embryonic and larval stages and are specifically involved in clotting reactions. Finally, lamellocytes are a special hemocyte cell-type that differentiates in response to infection by parasitic wasps and are required to encapsulate the invading organism to inhibit infection. Flies do not have a lymphocyte-like cell-type and do not show obvious signs of immunological memory, and as a result this model is only used to understand innate immune responses. However, the ability to live image hemocytes within this model, coupled with the evolutionary conservation of a number of immune signaling pathways makes this a powerful model to understand innate mechanisms behind wound-induced inflammation.
http://dx.doi.org/10.1016/j.smim.2014.04.004 1044-5323/© 2014 Elsevier Ltd. All rights reserved.
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2. Modeling epidermal wound recruitment and resolution in flies Drosophila has an open circulatory system and lacks blood vessels, therefore diapedesis during inflammation cannot be addressed in this model. However, plasmatocytes show very rapid responses to tissue damage and as a result this model has become a powerful system to dissect the innate signals that recruit inflammatory cells. Interestingly, depending on the developmental stage of the animal, the cellular response to damage is different, which has allowed for unique aspects of the response to be dissected. This section along with the next, will discuss Drosophila inflammatory responses during “epidermal” damage; however, it should be made clear that flies do not have a classical “epi”dermis as the outer epithelium in flies – which is also not stratified like the mammalian epidermis – lacks an underlying connective tissue layer i.e. a dermis. Therefore, tissue fibrosis, which is largely caused by dermal fibroblasts, cannot be modeled in Drosophila. Nevertheless, as we will see, a number of evolutionarily conserved innate immune responses during tissue repair can indeed be dissected in this simple organism. During embryogenesis, Drosophila hemocytes develop from precursor cells in the head mesoderm and spread throughout the embryo, appearing to take defined migratory routes [3]. Once they have dispersed they maintain an evenly spaced pattern through contact inhibition of locomotion [4,5] and patrol the hemocoel clearing up apoptotic debris as a result of normal embryogenesis. However, this dispersal pattern can be disrupted by tissue damage: induction of a wound by laser ablation to the embryonic epithelium leads to rapid recruitment of plasmatocytes to the wound site [6–9]. The hemocytes subsequently cease their contact inhibitory behavior and clump within the wound where they engulf cellular debris as a result of the damage. Over the next few hours (depending on the size of the wound), the epithelium will heal and plasmatocytes will return to circulation [8] suggesting that resolution of inflammation can also be modeled in this system (Fig. 1). After embryogenesis, flies enter larval stages where hemocyte behavior changes dramatically. While plasmatocytes are highly motile in embryos, larval plasmatocytes (which descend from embryonic hemocytes) transform into a completely sessile population and either adhere to tissues or passively circulate throughout the animal within the hemolymph. Plasmatocytes at larval stages, while not migratory, are still capable of recruitment to wounds. For example, damage to the epidermis leads to plasmatocyte recruitment to the wound through a “passive” mechanism whereby circulating cells adhere and become captured at the wound site [10]. Subsequently, as the epidermal wound heals, plasmatocytes return to circulation as the inflammation resolves. However, neither the capture nor resolution of inflammation within larval stages involves active cellular migration. There is speculation that plasmatocytes adhere specifically to the damaged basement membrane [11] and these cells may therefore express receptors that specifically recognize damaged matrix. Upon completion of larval stages, hemocyte behaviors change yet again. In pupae, the larval tissues histolyse and are replaced by adult populations of cells. Possibly to help clean up the debris as a result of the cell death, plasmatocyte populations become active, disperse throughout the animal, and re-acquire their capacity to actively migrate to wounds [12,13]. Recent work revealed that activation of hemocytes at pupal stages involves ecdysone, a steroid-like hormone in the fly, which drives the expression of plasmatocyte migratory genes [14]. This hormonal signaling is also required for plasmatocyte migratory responses to wounds [14]. It is currently unclear whether similar hormonal changes are responsible for the different behaviors of embryonic and larval hemocytes.
Fig. 1. Timecourse of hemocyte recruitment to embryonic epithelial wounds. (A) Drosophila embryo with a labeled epithelium (green) and hemocytes (magenta) wounded by laser ablation. Note that hemocytes accumulate within the epithelial wound site and subsequently disperse during the repair process. (B) Schematic showing the temporal relationship between hemocyte numbers at a wound and size of the damage. Note that upon peak recruitment of hemocytes, numbers at the wound site decrease in relation with the wound size suggesting the inflammation recruitment and resolution can be modeled in this system.
It is interesting that steroid hormones also affect mammalian leukocyte behaviors and inflammatory responses [15,16], although it remains to be determined whether their mechanisms of action are conserved in flies and vertebrates. Finally, it is intriguing to note that the migratory behaviors of adult hemocytes are completely unknown. The adult population is also immune responsive and capable of responding to infections, however it is unclear whether these cells are capable of actively migrating to sites of damage.
2.1. The role of Drosophila immune cells during epidermal repair The next question is, what is the function of hemocytes at wound sites? These cells are highly phagocytic, and within wounds – whether embryonic or larval – they will become massively swollen as they engulf large amounts of cellular debris [6,10]. However, similar to PU.1 knockout mice, which lack macrophages and neutrophils, flies lacking professional phagocytes are completely capable of healing epithelial wounds [6,10,17]. Similarly, in a regenerative model of repair in the developing Drosophila wing, hemocytes are dispensable [18]. This does not mean that there is no role for professional phagocytes at wound sites. As we will see, wound signals, even from sterile wounds, are capable of inducing anti-bacterial responses, as a major role of inflammation during repair is to protect against an increased risk of infection caused by a break in the epidermal barrier.
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2.2. Dissecting the signals behind Drosophila immune cell recruitment and resolution The rapidity with which inflammatory cells respond to wounds suggests that the initial recruitment signals are likely to be endogenous factors released from damaged or dying cells [19,20]. Indeed, within minutes of epithelial damage, embryonic plasmatocytes will respond and migrate to the wound site [6] suggesting that the recruiting signal is not the result of de novo production of some factor. While there are a host of endogenous proteins and chemicals that have been found capable of recruiting innate mammalian immune cells, it is currently unclear whether they are playing a role in Drosophila inflammation. However, one rapidly produced wound signal that has recently been found to be playing a role in wound responses in a number of species, including flies [8,9], is hydrogen peroxide [21]. Hydrogen peroxide release from damaged tissues and subsequent recruitment of inflammatory cells was first discovered in zebrafish, however, it was unclear exactly how it was being produced. Recently, Razzell et al., showed that damaged embryonic epithelial cells, similar to zebrafish, produce hydrogen peroxide through activation of an NADPH oxidase enzyme, DUOX. They went on to show that the rapidity of the hydrogen peroxide release was the result of induction of calcium waves surrounding the wound site as the DUOX enzyme is regulated by calcium [9,22]. It is currently unclear what is upstream of calcium activation at the wound site, or how hemocytes sense the hydrogen peroxide; however, the power of Drosophila genetics will certainly help to elucidate these questions. Drosophila as a wound model may also prove to be useful in understanding the resolution phase of inflammation. Work in mammalian systems has revealed that resolution of inflammation is not entirely passive as previously believed, but rather there are a number of active regulators that serve to dampen down the response [23]. Drosophila is also capable inflammation resolution during repair [8], however the mechanisms involved are currently unknown. It is interesting to speculate that plasmatocyte behavior may be actively modulated during the repair response. For example, “activated” plasmatocytes during embryonic repair must be capable of turning off their capacity for contact inhibition in order to congregate at wound sites, and they appear to turn this behavior back on upon returning to circulation when healing is complete [4,6,8]. It will be interesting to determine whether this behavior is actively controlled during wound repair and if vertebrate inflammatory cells exhibit a similar modulation of contact inhibitory behaviors.
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Indeed, many of the molecular mechanisms behind these glial responses have been elucidated in flies. The Drosophila gene draper, which is an ortholog of the C.elegans cell corpse engulfment gene, ced-1, is responsible for glial recognition of neuronal injury, and in flies mutated for draper, glia fail to undergo the morphological changes typical of the response to neuronal severing [29]. Again, the power of Drosophila genetics has allowed for the elucidation of the signaling pathways responsible for recognition of neuronal injury and subsequent engulfment of debris. Interestingly, draper is necessary for glial recognition of the initial damage cue (which is still unknown) and requires the tyrosine kinases Src42a and Shark [30,31]. In contrast, a completely distinct signaling cascade (involving the genes Crk, Mbc, and dCed12) is required to induce an engulfment program within glia at later stages of the wound response leading to phagocytosis of the damaged neuronal debris [32]. This data suggests that glial recruitment signals can be uncoupled from what appears to be a distinct engulfment cascade. It remains to be determined whether an identical program of events is required for activation of mammalian glial cells during neuronal injury. It will also be interesting to determine whether there are tissue repair immune responses conserved across tissue types. Draper contains an immunoreceptor tyrosinebased activation motif (ITAM), which has emerged as a motif common to many receptors critical for modulating mammalian immune responses in a variety of inflammatory cells [33,34], and it is interesting to speculate that Draper may be playing a more general role in inducing inflammatory responses in flies. Furthermore, evolutionarily conserved components of the mammalian AP-1 transcription factor, Drosophila Jra and Kayak, are also involved in glial activation [35]; as AP-1 is a well-established early signaling mediator in mammalian skin repair [36,37], the Drosophila CNS injury model may indeed be informative for elucidating conserved wound healing responses across both species and tissue types. 4. Dissecting systemic wound signals The response to tissue damage is not restricted to the local wound area: in flies as in vertebrates, wounds cause the production of a variety of soluble signals that can act on quite distant tissues, which leads to a systemic wound response [38–40]. Several recent papers have begun to decipher these effects on distant tissues and the signals that regulate them. In this section, we will explore the systemic effects of wound-derived signals. 4.1. Wounds and distant effects on cellular immunity
3. Dissecting inflammatory reactions in the fly CNS Tissue repair is not solely a response in skin as every organ in the body is capable of healing to varying degrees. Due to distinct structural components of our organ systems, each tissue will elicit a unique reaction to damage (although some similarities will clearly exist), and therefore we must understand repair responses in each tissue type independently. One example of a highly studied organ with unique responses to tissue damage is the brain. Neuronal cells can be damaged during physical injury, or as a result of a number of different CNS pathologies. Regardless of the insult, damage results in rapid responses in surrounding glia [24–27], which are the support cells of the CNS. Drosophila glia exhibit many similarities with the four major types of glia found in mammals, including microglia which are derived from the monocyte/macrophage lineage [28]. After neuronal injury in both flies and mammals, surrounding glia undergo morphological and transcriptional changes, which allows them to rapidly clear the damaged neurons by phagocytosis [24,29].
Mechanical damage to larval tissues is capable of inducing an increase in the number of circulating hemocytes via a global induction of hemocyte proliferation. This response is induced via local JNK activation within damaged tissues, which subsequently leads to systemic cytokine production and activation of JAK/STAT signals within hemocytes. Furthermore, the hemocytes themselves also produce the cytokine, suggesting that this proliferative response is the result of an autocrine positive feedback loop [11]. Larval wounds also induce a systemic response that regulates the larval hematopoietic program. In larvae that are not wounded, the lymph gland (the larval hematopoietic organ) contains prohemocytes that differentiate within the organ into plasmatocytes. Under normal circumstances (i.e. In the absence of a wound) at the end of larval life the lymph gland bursts, and the released plasmatocytes subsequently assist in clearing larval tissues to permit the generation of the adult fly. However, wounding diverts this prohemocyte population to produce lamellocytes instead of plasmatocytes. Lamellocytes are large, sheet-like cells that are able to encapsulate invading organisms too large to be phagocytosed;
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they are a critical element in the immune response against parasitoid wasps, which lay their eggs in the larvae of other insects. The wasp’s ovipositor must breach the larval cuticle when inserting an egg and this sterile wound is sufficient to trigger lamellocyte differentiation [41]. The wound-derived signal that triggers this differentiation event is still unknown. One plausible candidate is wound-derived reactive oxygen species signaling to the lymph gland posterior signaling center, which would in turn signal via an epidermal growth factor receptor (EGFR) pathway to drive lamellocyte differentiation in prohemocytes [42]. Sterile wounds in the adult fly also induce a systemic wound response. Sterile wounds to the integument activates a serine protease cascade, which leads to redox-dependent activation of JNK in distant neuronal tissues. Specific inhibition of ROS signaling after wounding leads to enhanced lethality, suggesting that this cascade is cytoprotective during the wound response [43]. It is interesting that the protease cascade required for this ROS induction is also involved in humoral immunity, which suggests a link between sterile wounds and infection responses (see Section 4.2). 4.2. Wounds and the humoral immune response In addition to the cellular response, Drosophila exhibit a potent humoral response to infection. Humoral immunity in the fly is characterized by the rapid transcriptional induction of many soluble secreted antimicrobial factors, including lysozymes and antimicrobial peptides (AMPs). This is primarily dependent on the Toll and imd signaling pathways. Each pathway responds to distinct microbial elicitors and culminates in activation of one or more NFB family transcription factors. These pathways can be activated locally, at barrier epithelia, or systemically, in the fat body [44–47]. Though microbial elicitors are required for maximal induction of Toll and imd pathways, apparently sterile wounding is sufficient to induce AMP expression to a detectable extent [48–50]. This is true in our hands even of wounds inflicted without any breach in the cuticle, preventing any possibility of cryptic bacterial infection; this induction is dependent on the imd pathway (unpublished data, Dionne lab). It is not known where, anatomically, this induction takes place, though prior work on infected wounds in Drosophila larvae suggests strongly that the wound-site epithelium, rather than the fat body, is the responsible tissue [47]. Sterile wounds induce expression of two TGF- superfamily members, decapentaplegic (dpp) and dawdle [48]. Both these signals appear to come, at least in part, from plasmatocytes. The induced dpp signal is received by the fat body, where it transcriptionally down-regulates expression of AMPs. Flies in which the ability of the fat body to respond to this signal is abrogated are much more sensitive to wounding than wild-type animals, tending to die within a week of receiving even a small wound (wild-type animals exhibit no such lethality) (unpublished data, Dionne lab). It is not clear whether this death is due to the increased AMP expression exhibited by these animals. However, it is clear that an uncontrolled systemic wound response is detrimental to Drosophila survival [51]. Nevertheless, this data suggests that, similar to mammals, TGF- signals are broadly anti-inflammatory in nature [52,53]. 5. Conclusion While the Drosophila system may not be the ideal model to understand all aspects of immune responses during wounding, there are facets of this process that are ideally addressed in this basic organism. The ability to image Drosophila immune responses at high spatial and temporal resolution is starting to help us elucidate the most upstream signals that are responsible for
initiating the repair process, which have largely been a mystery until recently. The genetic tractability of flies, and the relative lack of genetic redundancy, allows us to dissect the precise genes and signaling cascades responsible for both local and systemic wound responses. The final reason to utilize such a basic model organism is fortuitous in that we gain a knowledge about evolutionarily conserved responses, which will highlight critical players in the immune response to wounds that will likely be clinically significant.
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Review
Unraveling tissue repair immune responses in flies Brian M. Stramer a,∗ , Marc S. Dionne b a b
King’s College London, Randall Division of Cell and Molecular Biophysics, London SE1 1UL, United Kingdom King’s College London, Centre for the Molecular and Cellular Biology of Inflammation, London SE1 1UL, United Kingdom
a r t i c l e
i n f o
Keywords: Drosophila Wound repair
a b s t r a c t Drosophila melanogaster has emerged as a powerful model to understand innate immune responses to infection (note the 2011 Nobel Prize in Physiology or Medicine), and in recent years this system has begun to inform on the role and regulation of immune responses during tissue injury. Due to the speed and complexity of inflammation signals upon damage, a complete understanding of the immune responses during repair requires a combination of live imaging at high temporal resolution and genetic dissection, which is possible in a number of different injury models in the fly. Here we discuss the range of woundinduced immune responses that can be modeled in flies. These wound models have revealed the most immediate signals leading to immune cell activation, and highlighted a number of complex signaling cascades required for subsequent injury-associated inflammatory responses. What has emerged from this system are a host of both local acting signals, and surprisingly, more systemic tissue repair immune responses. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Most of what we know about immune responses during tissue damage has been gleaned from studies of human tissues and cells, or from mammalian models. However, the complexity of inflammation, and its dynamic nature make it problematic to dissect at a genetic level and difficult to live image. As a result, a number of basic model organisms are starting to be exploited. Ilya Metchnikov was the first to utilize a model organism, starfish larvae, to observe the inflammatory response to wounding [1]. While a simple organism like starfish, does not exhibit the Oxford English Dictionary’s definition of inflammation – rubor (redness), calor (heat), tumor (swelling), and dolor (pain) – the central etiology of its wound inflammation (which was highlighted in Ilya’s Nobel lecture) is the same: recruitment of professional microbe killing and debris clearing cells. Indeed, Drosophila, which is far more genetically tractable than Metchnikov’s starfish, has been used as a model of animal immunity for some time. Strikingly, key components of a Drosophila’s response to pathogenic infection, such as a requirement for Toll signaling, are evolutionarily conserved (and in 2011 a Nobel Prize in Physiology or Medicine was awarded for this discovery). While most of the Drosophila work has focused on
∗ Corresponding author. Tel.: +44 0207 848 6272. E-mail addresses: [email protected] (B.M. Stramer), [email protected] (M.S. Dionne).
infection responses, flies are increasingly being utilized to understand the role and regulation of immune responses during wound healing. This review will discuss the physiologically relevant questions that can be addressed in this model, and highlight recent discoveries that have exploited the benefits of this basic model system. 1.1. Drosophila immune cells Flies have a relatively simple immune system that is composed of hemocytes (insect “blood” cells) that can differentiate into just a few cell-types [2]. The major hemocyte population is the plasmatocyte, which is a macrophage-like phagocytic cell-type that is capable of migratory responses to tissue damage. In contrast, Crystal cells make up a small percentage of the total hemocyte population at embryonic and larval stages and are specifically involved in clotting reactions. Finally, lamellocytes are a special hemocyte cell-type that differentiates in response to infection by parasitic wasps and are required to encapsulate the invading organism to inhibit infection. Flies do not have a lymphocyte-like cell-type and do not show obvious signs of immunological memory, and as a result this model is only used to understand innate immune responses. However, the ability to live image hemocytes within this model, coupled with the evolutionary conservation of a number of immune signaling pathways makes this a powerful model to understand innate mechanisms behind wound-induced inflammation.
http://dx.doi.org/10.1016/j.smim.2014.04.004 1044-5323/© 2014 Elsevier Ltd. All rights reserved.
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2. Modeling epidermal wound recruitment and resolution in flies Drosophila has an open circulatory system and lacks blood vessels, therefore diapedesis during inflammation cannot be addressed in this model. However, plasmatocytes show very rapid responses to tissue damage and as a result this model has become a powerful system to dissect the innate signals that recruit inflammatory cells. Interestingly, depending on the developmental stage of the animal, the cellular response to damage is different, which has allowed for unique aspects of the response to be dissected. This section along with the next, will discuss Drosophila inflammatory responses during “epidermal” damage; however, it should be made clear that flies do not have a classical “epi”dermis as the outer epithelium in flies – which is also not stratified like the mammalian epidermis – lacks an underlying connective tissue layer i.e. a dermis. Therefore, tissue fibrosis, which is largely caused by dermal fibroblasts, cannot be modeled in Drosophila. Nevertheless, as we will see, a number of evolutionarily conserved innate immune responses during tissue repair can indeed be dissected in this simple organism. During embryogenesis, Drosophila hemocytes develop from precursor cells in the head mesoderm and spread throughout the embryo, appearing to take defined migratory routes [3]. Once they have dispersed they maintain an evenly spaced pattern through contact inhibition of locomotion [4,5] and patrol the hemocoel clearing up apoptotic debris as a result of normal embryogenesis. However, this dispersal pattern can be disrupted by tissue damage: induction of a wound by laser ablation to the embryonic epithelium leads to rapid recruitment of plasmatocytes to the wound site [6–9]. The hemocytes subsequently cease their contact inhibitory behavior and clump within the wound where they engulf cellular debris as a result of the damage. Over the next few hours (depending on the size of the wound), the epithelium will heal and plasmatocytes will return to circulation [8] suggesting that resolution of inflammation can also be modeled in this system (Fig. 1). After embryogenesis, flies enter larval stages where hemocyte behavior changes dramatically. While plasmatocytes are highly motile in embryos, larval plasmatocytes (which descend from embryonic hemocytes) transform into a completely sessile population and either adhere to tissues or passively circulate throughout the animal within the hemolymph. Plasmatocytes at larval stages, while not migratory, are still capable of recruitment to wounds. For example, damage to the epidermis leads to plasmatocyte recruitment to the wound through a “passive” mechanism whereby circulating cells adhere and become captured at the wound site [10]. Subsequently, as the epidermal wound heals, plasmatocytes return to circulation as the inflammation resolves. However, neither the capture nor resolution of inflammation within larval stages involves active cellular migration. There is speculation that plasmatocytes adhere specifically to the damaged basement membrane [11] and these cells may therefore express receptors that specifically recognize damaged matrix. Upon completion of larval stages, hemocyte behaviors change yet again. In pupae, the larval tissues histolyse and are replaced by adult populations of cells. Possibly to help clean up the debris as a result of the cell death, plasmatocyte populations become active, disperse throughout the animal, and re-acquire their capacity to actively migrate to wounds [12,13]. Recent work revealed that activation of hemocytes at pupal stages involves ecdysone, a steroid-like hormone in the fly, which drives the expression of plasmatocyte migratory genes [14]. This hormonal signaling is also required for plasmatocyte migratory responses to wounds [14]. It is currently unclear whether similar hormonal changes are responsible for the different behaviors of embryonic and larval hemocytes.
Fig. 1. Timecourse of hemocyte recruitment to embryonic epithelial wounds. (A) Drosophila embryo with a labeled epithelium (green) and hemocytes (magenta) wounded by laser ablation. Note that hemocytes accumulate within the epithelial wound site and subsequently disperse during the repair process. (B) Schematic showing the temporal relationship between hemocyte numbers at a wound and size of the damage. Note that upon peak recruitment of hemocytes, numbers at the wound site decrease in relation with the wound size suggesting the inflammation recruitment and resolution can be modeled in this system.
It is interesting that steroid hormones also affect mammalian leukocyte behaviors and inflammatory responses [15,16], although it remains to be determined whether their mechanisms of action are conserved in flies and vertebrates. Finally, it is intriguing to note that the migratory behaviors of adult hemocytes are completely unknown. The adult population is also immune responsive and capable of responding to infections, however it is unclear whether these cells are capable of actively migrating to sites of damage.
2.1. The role of Drosophila immune cells during epidermal repair The next question is, what is the function of hemocytes at wound sites? These cells are highly phagocytic, and within wounds – whether embryonic or larval – they will become massively swollen as they engulf large amounts of cellular debris [6,10]. However, similar to PU.1 knockout mice, which lack macrophages and neutrophils, flies lacking professional phagocytes are completely capable of healing epithelial wounds [6,10,17]. Similarly, in a regenerative model of repair in the developing Drosophila wing, hemocytes are dispensable [18]. This does not mean that there is no role for professional phagocytes at wound sites. As we will see, wound signals, even from sterile wounds, are capable of inducing anti-bacterial responses, as a major role of inflammation during repair is to protect against an increased risk of infection caused by a break in the epidermal barrier.
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2.2. Dissecting the signals behind Drosophila immune cell recruitment and resolution The rapidity with which inflammatory cells respond to wounds suggests that the initial recruitment signals are likely to be endogenous factors released from damaged or dying cells [19,20]. Indeed, within minutes of epithelial damage, embryonic plasmatocytes will respond and migrate to the wound site [6] suggesting that the recruiting signal is not the result of de novo production of some factor. While there are a host of endogenous proteins and chemicals that have been found capable of recruiting innate mammalian immune cells, it is currently unclear whether they are playing a role in Drosophila inflammation. However, one rapidly produced wound signal that has recently been found to be playing a role in wound responses in a number of species, including flies [8,9], is hydrogen peroxide [21]. Hydrogen peroxide release from damaged tissues and subsequent recruitment of inflammatory cells was first discovered in zebrafish, however, it was unclear exactly how it was being produced. Recently, Razzell et al., showed that damaged embryonic epithelial cells, similar to zebrafish, produce hydrogen peroxide through activation of an NADPH oxidase enzyme, DUOX. They went on to show that the rapidity of the hydrogen peroxide release was the result of induction of calcium waves surrounding the wound site as the DUOX enzyme is regulated by calcium [9,22]. It is currently unclear what is upstream of calcium activation at the wound site, or how hemocytes sense the hydrogen peroxide; however, the power of Drosophila genetics will certainly help to elucidate these questions. Drosophila as a wound model may also prove to be useful in understanding the resolution phase of inflammation. Work in mammalian systems has revealed that resolution of inflammation is not entirely passive as previously believed, but rather there are a number of active regulators that serve to dampen down the response [23]. Drosophila is also capable inflammation resolution during repair [8], however the mechanisms involved are currently unknown. It is interesting to speculate that plasmatocyte behavior may be actively modulated during the repair response. For example, “activated” plasmatocytes during embryonic repair must be capable of turning off their capacity for contact inhibition in order to congregate at wound sites, and they appear to turn this behavior back on upon returning to circulation when healing is complete [4,6,8]. It will be interesting to determine whether this behavior is actively controlled during wound repair and if vertebrate inflammatory cells exhibit a similar modulation of contact inhibitory behaviors.
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Indeed, many of the molecular mechanisms behind these glial responses have been elucidated in flies. The Drosophila gene draper, which is an ortholog of the C.elegans cell corpse engulfment gene, ced-1, is responsible for glial recognition of neuronal injury, and in flies mutated for draper, glia fail to undergo the morphological changes typical of the response to neuronal severing [29]. Again, the power of Drosophila genetics has allowed for the elucidation of the signaling pathways responsible for recognition of neuronal injury and subsequent engulfment of debris. Interestingly, draper is necessary for glial recognition of the initial damage cue (which is still unknown) and requires the tyrosine kinases Src42a and Shark [30,31]. In contrast, a completely distinct signaling cascade (involving the genes Crk, Mbc, and dCed12) is required to induce an engulfment program within glia at later stages of the wound response leading to phagocytosis of the damaged neuronal debris [32]. This data suggests that glial recruitment signals can be uncoupled from what appears to be a distinct engulfment cascade. It remains to be determined whether an identical program of events is required for activation of mammalian glial cells during neuronal injury. It will also be interesting to determine whether there are tissue repair immune responses conserved across tissue types. Draper contains an immunoreceptor tyrosinebased activation motif (ITAM), which has emerged as a motif common to many receptors critical for modulating mammalian immune responses in a variety of inflammatory cells [33,34], and it is interesting to speculate that Draper may be playing a more general role in inducing inflammatory responses in flies. Furthermore, evolutionarily conserved components of the mammalian AP-1 transcription factor, Drosophila Jra and Kayak, are also involved in glial activation [35]; as AP-1 is a well-established early signaling mediator in mammalian skin repair [36,37], the Drosophila CNS injury model may indeed be informative for elucidating conserved wound healing responses across both species and tissue types. 4. Dissecting systemic wound signals The response to tissue damage is not restricted to the local wound area: in flies as in vertebrates, wounds cause the production of a variety of soluble signals that can act on quite distant tissues, which leads to a systemic wound response [38–40]. Several recent papers have begun to decipher these effects on distant tissues and the signals that regulate them. In this section, we will explore the systemic effects of wound-derived signals. 4.1. Wounds and distant effects on cellular immunity
3. Dissecting inflammatory reactions in the fly CNS Tissue repair is not solely a response in skin as every organ in the body is capable of healing to varying degrees. Due to distinct structural components of our organ systems, each tissue will elicit a unique reaction to damage (although some similarities will clearly exist), and therefore we must understand repair responses in each tissue type independently. One example of a highly studied organ with unique responses to tissue damage is the brain. Neuronal cells can be damaged during physical injury, or as a result of a number of different CNS pathologies. Regardless of the insult, damage results in rapid responses in surrounding glia [24–27], which are the support cells of the CNS. Drosophila glia exhibit many similarities with the four major types of glia found in mammals, including microglia which are derived from the monocyte/macrophage lineage [28]. After neuronal injury in both flies and mammals, surrounding glia undergo morphological and transcriptional changes, which allows them to rapidly clear the damaged neurons by phagocytosis [24,29].
Mechanical damage to larval tissues is capable of inducing an increase in the number of circulating hemocytes via a global induction of hemocyte proliferation. This response is induced via local JNK activation within damaged tissues, which subsequently leads to systemic cytokine production and activation of JAK/STAT signals within hemocytes. Furthermore, the hemocytes themselves also produce the cytokine, suggesting that this proliferative response is the result of an autocrine positive feedback loop [11]. Larval wounds also induce a systemic response that regulates the larval hematopoietic program. In larvae that are not wounded, the lymph gland (the larval hematopoietic organ) contains prohemocytes that differentiate within the organ into plasmatocytes. Under normal circumstances (i.e. In the absence of a wound) at the end of larval life the lymph gland bursts, and the released plasmatocytes subsequently assist in clearing larval tissues to permit the generation of the adult fly. However, wounding diverts this prohemocyte population to produce lamellocytes instead of plasmatocytes. Lamellocytes are large, sheet-like cells that are able to encapsulate invading organisms too large to be phagocytosed;
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they are a critical element in the immune response against parasitoid wasps, which lay their eggs in the larvae of other insects. The wasp’s ovipositor must breach the larval cuticle when inserting an egg and this sterile wound is sufficient to trigger lamellocyte differentiation [41]. The wound-derived signal that triggers this differentiation event is still unknown. One plausible candidate is wound-derived reactive oxygen species signaling to the lymph gland posterior signaling center, which would in turn signal via an epidermal growth factor receptor (EGFR) pathway to drive lamellocyte differentiation in prohemocytes [42]. Sterile wounds in the adult fly also induce a systemic wound response. Sterile wounds to the integument activates a serine protease cascade, which leads to redox-dependent activation of JNK in distant neuronal tissues. Specific inhibition of ROS signaling after wounding leads to enhanced lethality, suggesting that this cascade is cytoprotective during the wound response [43]. It is interesting that the protease cascade required for this ROS induction is also involved in humoral immunity, which suggests a link between sterile wounds and infection responses (see Section 4.2). 4.2. Wounds and the humoral immune response In addition to the cellular response, Drosophila exhibit a potent humoral response to infection. Humoral immunity in the fly is characterized by the rapid transcriptional induction of many soluble secreted antimicrobial factors, including lysozymes and antimicrobial peptides (AMPs). This is primarily dependent on the Toll and imd signaling pathways. Each pathway responds to distinct microbial elicitors and culminates in activation of one or more NFB family transcription factors. These pathways can be activated locally, at barrier epithelia, or systemically, in the fat body [44–47]. Though microbial elicitors are required for maximal induction of Toll and imd pathways, apparently sterile wounding is sufficient to induce AMP expression to a detectable extent [48–50]. This is true in our hands even of wounds inflicted without any breach in the cuticle, preventing any possibility of cryptic bacterial infection; this induction is dependent on the imd pathway (unpublished data, Dionne lab). It is not known where, anatomically, this induction takes place, though prior work on infected wounds in Drosophila larvae suggests strongly that the wound-site epithelium, rather than the fat body, is the responsible tissue [47]. Sterile wounds induce expression of two TGF- superfamily members, decapentaplegic (dpp) and dawdle [48]. Both these signals appear to come, at least in part, from plasmatocytes. The induced dpp signal is received by the fat body, where it transcriptionally down-regulates expression of AMPs. Flies in which the ability of the fat body to respond to this signal is abrogated are much more sensitive to wounding than wild-type animals, tending to die within a week of receiving even a small wound (wild-type animals exhibit no such lethality) (unpublished data, Dionne lab). It is not clear whether this death is due to the increased AMP expression exhibited by these animals. However, it is clear that an uncontrolled systemic wound response is detrimental to Drosophila survival [51]. Nevertheless, this data suggests that, similar to mammals, TGF- signals are broadly anti-inflammatory in nature [52,53]. 5. Conclusion While the Drosophila system may not be the ideal model to understand all aspects of immune responses during wounding, there are facets of this process that are ideally addressed in this basic organism. The ability to image Drosophila immune responses at high spatial and temporal resolution is starting to help us elucidate the most upstream signals that are responsible for
initiating the repair process, which have largely been a mystery until recently. The genetic tractability of flies, and the relative lack of genetic redundancy, allows us to dissect the precise genes and signaling cascades responsible for both local and systemic wound responses. The final reason to utilize such a basic model organism is fortuitous in that we gain a knowledge about evolutionarily conserved responses, which will highlight critical players in the immune response to wounds that will likely be clinically significant.
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