Insects, including the fruit fly, Drosophila melanogaster are used to study a wide array of processes, many of which are known or are expected to be regulated by damage-associated molecular patterns (DAMPs). These include regenerative processes after wounding, replacement of cells by cell competition, induction of immunity and inflammation, responses against tumorous cells and neurodegeneration. Most, if not all of these processes have beneficial outcomes on organismal health but may also lead to pathologies, which often resemble those observed in humans. Drosophila offers unique opportunities to analyze and manipulate genes and pathways related to these immune consequences with high temporal and local resolution. Ultimately, such detailed analyses in the Drosophila model will aid in our understanding of the roles DAMPs play at the bifurcation between physiological and pathological outcomes in other animal species, including humans.
Keywords: Coagulation, Danger signals, DAMPs, Hemocytes, Inflammation, Innate immunity, Insect immunity, Regeneration, Tumors, Wound healing
DROSOPHILA IMMUNITY - A QUICK GUIDE
WOUND SEALING AND HEALING: LIVE IMAGING AND BEYOND
In an infection context, hemolymph clots have been demonstrated to prevent entry of parasites that target epithelial surfaces using mechanical tools such as entomopathogenic nematodes (EPNs), which use their mouth part to gain entry to the hemocoel . EPN infection leads to a massive induction of immune-related genes although some immune genes appear to depend on the clot or clot components for their induction rather than on microbial or parasite-specific elicitors  and thus are more akin to microbe-independent responses like those observed during sterile inflammation. In microarray data of Drosophila infected with the EPN Heterorhabditis bacteriophora, it was found that several hundred genes are specifically induced in EPN infection in comparison to other types of infections, like parasitic wasp infection, and that there are several candidate damage-induced molecules such as thioester containing protein-1, Eiger, Spätzle-processing enzyme and potentially others that are yet to be characterized [17,18]. Interestingly, the Toll reporter Drosomycin is highest on that list providing evidence for alternative ways of inducing AMPs independent of the Toll pathway. Similarly, Hauling et al. , through RNA sequencing of the fat body gained further insights into the endogenous response against danger signals produced from tumor-expressing salivary glands . Beyond the current molecular scope, we have successfully been implementing the use of sequencing data as a map for finding new danger signals and DAMPs.
Released DAMPs enable broad trans-tissue communication. In external wounding situations, local DAMPs at wound sites establish local communication between the wound and the immune system to ensure that a proper wound healing response is taking place. In injured organs, actin can be exposed on the surface of the wound site . Similar findings have revealed that actin is also found in the blood, suggesting that it may be released from injured tissue . However, the mechanism of DAMP-actin-induced inflammation is not fully understood. Another role actin may have is to serve as a signal molecule . For instance, Ahrens et al.  found that actin is a danger signal that is conserved from yeast to humans. F-actin acts as a ligand and is recognized by a DAMP receptor for dead cells called DNGR-1 (also known as CLEC9A) in both vertebrates and Saccharomyces cerevisiae . In mosquitoes, actin has been found to promote phagocytosis of bacteria in cooperation with the small MD2-like protein and to act as a Plasmodium falciparum antagonist . Recently, Srinivasan et al.  identified actin as a conserved DAMP in the fruit fly. Following injection of actin into Drosophila larvae, they observed induced sterile inflammation in the fat body. They identified that exogenous actin acts as a conserved signal that is released from damaged cells which leads to a selective JAK/STAT response . While the actual actin receptor remains elusive (there is no fly homologue for the mammalian actin receptor DNGR-1 and Draper appears dispensable), the actin signal appears to feed into the SFK-Draper-Shark pathway further strengthening its evolutionary conservation as a cell injury detector which precedes the evolution of adaptive immunity.
NON-CANONICAL TOLL SIGNALING: A VERSATILE TOOL 
PERSEPHONE: BAIT FOR EXOGENOUS PROTEASES AND ENDOGENOUS SIGNALS
In trained immunity in mammals, innate immune cells can be primed by primary infections or vaccination to perform more efficiently upon subsequent exposure to microbial attack. Similarly, insect immunity can be primed by previous exposure to antigens, a phenomenon that has been dubbed “immune memory” or “immune priming” . While trained immunity in mammals confers broad range protection against unspecific microbes, at least some cases of insect immune priming appear to be quite specific . Though the possibility that the mechanism exists in vertebrates and organisms with an adaptive immunity has not yet been ruled out. While exogenous signals have been found responsible for immune training/priming, similarly in mammals and insects, tissue damage appears to play a central educational role for insect hemocytes . Like macrophages, hemocytes are multifunctional cells taking care of both internal damage and microbial attack by for example, phagocytosing bacteria or removing apoptotic cells. During the removal of apoptotic cells, Weavers et al.  showed that Drosophila embryonic development is essential for priming hemocytes both to efficiently perform wound healing and to fight infections. Priming is triggered by calcium flashes which activate JNK signaling and subsequent induction of the apoptotic regulator Draper, a key molecule in wound healing. Consequently, inhibiting apoptosis as well as interfering with JNK signaling affects the inflammatory potentials of hemocytes, which is somewhat expected but surprisingly, their immune competence is hindered for example, they lose the ability to phagocytose Escherichia coli . Trained immunity has also been demonstrated in the case of viral infections in the fruit fly. Cellular damage releases viral dsRNA which is subsequently phagocytised by plasmatocytes and eventually packaged into endosomes to transfer antiviral RNAi to other hemocytes . Thus the mechanism for priming hemocytes for a specific viral infection initially bears the hallmarks of damage-induced clearance but leads to specific protection. This varies widely from what has thus far been shown in the adaptive immune system in the mammalian model, but demonstrates that the innate immune system can play a similar role in immune priming.
CANCER AND DANGER: A MULTILEVEL AFFAIR
DANGER IN THE NERVOUS SYSTEM: NEUROINFLAMMATION
A number of approaches have been used to induce damage in the fly’s nervous system (NS) which include the expression of human disease-causing gene polymorphisms in the NS  as well as through causing mechanical damage . Upon severing axons that lead to either wings or legs, transcriptome profiling of the ventral nerve cord revealed two main pathways involved in other wounding scenarios, mainly Draper/AP-1 and Toll, were upregulated. In addition, Stat92E/draper/JNK/AP-1 activity was necessary for metalloproteinase-1 (MMP1) to successfully clear away debris of severed axons and for regeneration . While this study demonstrated activation of stress was required for innate glial immunity, another study used a fly model of Ataxia-Telangiectasia to demonstrate that the NFkB factor, Relish and the induction of select immune genes were key culprits for neurodegeneration  in a non-canonical manner (the imd pathway was dispensable). When different models of neurodegenerative scenarios are compared, an immune signature is often identified. It has been proposed that this immune signature may actually reveal an equally important neuroprotective function of the “immune genes” since both DAMPs and damage clearance play key roles during infections and tissue healing, a pleiotropy that is often misrepresented during gene annotation [50,51]. An example of this duality which exists within genes’ function is provided by a member of a prototypical PRR family (PGRP-LC, a peptidoglycan receptor in the Drosophila imd pathway), which is also required for synaptic plasticity in mice . Similarly, immune transcription factor isoforms have additional (including regenerative) functions in non-immune tissues [53,54].
CONCLUSION - DROSOPHILA OFFERS AN INTEGRATIVE VIEW OF IMMUNITY
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