| 1. |
- Arefin, Badrul, et al.
(författare)
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Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naive larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemo(less)) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll-as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.
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| 2. |
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| 3. |
- Arefin, Badrul, et al.
(författare)
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Genome-Wide Transcriptional Analysis of Drosophila Larvae Infected by Entomopathogenic Nematodes Shows Involvement of Complement, Recognition and Extracellular Matrix Proteins
- 2014
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 6:2, s. 192-204
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Tidskriftsartikel (refereegranskat)abstract
- Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.
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| 4. |
- Arefin, Badrul, et al.
(författare)
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The Immune Phenotype of Three Drosophila Leukemia Models
- 2017
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Ingår i: G3. - : Oxford University Press (OUP). - 2160-1836. ; 7:7, s. 2139-2149
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Tidskriftsartikel (refereegranskat)abstract
- Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruit fly Drosophila melanogaster has an innate immune system, including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, we established three models by driving expression of a dominant-active version of the Ras oncogene (Ras(V12)) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. Our results show that phagocytosis, hemocyte migration to wound sites, wound sealing, and survival upon bacterial infection of leukemic lines are similar to wild type. We find that in all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll-dependent signaling is activated to comparable extents as after wounding wild-type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, we notice that adult tissue formation is blocked and degradation of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic lines, we used a natural infection model that involves insect-pathogenic nematodes. We identified two leukemic lines that were sensitive to nematode infections. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together, this work establishes new Drosophila models to study the physiological, immunological, and behavioral consequences of various forms of leukemia.
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| 5. |
- Arefin, Md. Badrul, 1983-
(författare)
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Molecular characterization of the Drosophila responses towards nematodes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above.We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity.Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations.
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| 6. |
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| 7. |
- Bidla, Gawa, et al.
(författare)
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Activation of Insect Phenoloxidase after Injury : Endogenous versus Foreign Elicitors
- 2009
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 1:4, s. 301-308
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. We tested how PO is activated following injury in 2 insects, i.e. the fruit fly Drosophila melanogaster and the wax moth Galleria mellonella. Rapid PO activation in Drosophila was limited to discrete areas of the hemolymph clot which forms after injury. Surprisingly, unlike systemic PO activation during bacterial sepsis, clot melanization was not sensitive to microbial elicitors in our assay. Instead, Drosophila clot melanization was activated by endogenous signals such as apoptotic cells and was superinduced by phosphatidylserine, a negatively charged phospholipid normally found on the inner surface of the plasma membrane and exposed during apoptosis. In contrast, melanization in G. mellonella hemolymph was stronger and more uniform and was sensitive to peptidoglycan. This shows that both exogenous and endogenous signals can trigger the same immune mechanism in species and context-dependent ways. Our findings have implications for the evolutionary dynamics of immune mechanisms and are in agreement with recent comparisons of insect immune transcriptomes.
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| 8. |
- Bidla, Gawa, et al.
(författare)
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Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog eiger
- 2007
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 120:7, s. 1209-1215
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Tidskriftsartikel (refereegranskat)abstract
- The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.
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| 9. |
- Bidla, Gawa, 1975-
(författare)
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Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies. By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture. The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug. We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own.
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| 10. |
- Dobes, Pavel, et al.
(författare)
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An improved method for nematode infection assays in Drosophila larvae
- 2012
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Ingår i: Fly. - : Informa UK Limited. - 1933-6934 .- 1933-6942. ; 6:2, s. 75-79
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Tidskriftsartikel (refereegranskat)abstract
- The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) seek out host insects and release their symbiotic bacteria into their body cavity causing septicaemia, which eventually leads to host death. The interaction between EPNs and their hosts are only partially understood, in particular the host immune responses appears to involve pathways other than phagocytosis and the canonical transcriptional induction pathways. These pathways are genetically tractable and include for example clotting factors and lipid mediators. The aim of this study was to optimize the nematode infections inDrosophila melanogaster larvae, a well-studied and genetically tractable model organism. Here we show that two nematode species namely Steinernema feltiae and Heterorhabditis bacteriophora display different infectivity towards Drosophila larvae with the latter being less pathogenic. The effects of supporting media and IJ dosage on the mortality of the hosts were assessed and optimized. Using optimum conditions, a faster and efficient setup for nematode infections was developed. This newly established infection model in Drosophila larvae will be applicable in large scale screens aimed at identifying novel genes/pathways involved in innate immune responses.
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| 11. |
- Dziedziech, Alexis, 1991-, et al.
(författare)
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Convergent evolution of mammalian inflammatory cell death mechanism, pyroptosis in Drosophila melanogaster
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines upon cell bursting. This form of cell death has been increasingly well-characterized in mammals but there has been little evidence for the existence of pyroptosis across phyla. Here we provide evidence for convergent evolution of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to the wound and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects. Further, we unveil key players upstream in the activation of cell death in these cells., Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.
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| 12. |
- Dziedziech, Alexis, et al.
(författare)
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Data on Drosophila clots and hemocyte morphologies using GFP-tagged secretory proteins : Prophenoloxidase and transglutaminase
- 2019
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 25
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Tidskriftsartikel (refereegranskat)abstract
- Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors Schmid et al., 2019. The linked article demonstrates the localization of two secretory proteins in Drosophila melanogaster, Prophenoloxidase (PPO2) and Transglutaminase-A (Tg) in hemocytes as well the clot with different tissue-specific drivers. Here we provide further data for the usefulness of the GFP-tagged version of the two crosslinking enzymes that are involved in clot hardening. The morphology of crystal cells is described using GFP-tagged PPO2 rather than with the use of antibodies in ex vivo hemolymph preparations. The use of the GFP-tagged proteins PPO2 and Tg is shown in additional contexts.
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| 13. |
- Dziedziech, Alexis, et al.
(författare)
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Digging Back in Evolution : Danger in Drosophila
- 2018
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Ingår i: Journal of Damage-Associated Molecular Patterns. ; 1:1, s. 1-8
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 14. |
- Dziedziech, Alexis, et al.
(författare)
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Drosophila melanogaster Responses against Entomopathogenic Nematodes : Focus on Hemolymph Clots
- 2020
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Ingår i: Insects. - : MDPI AG. - 2075-4450. ; 11:1
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Forskningsöversikt (refereegranskat)abstract
- Several insect innate immune mechanisms are activated in response to infection by entomopathogenic nematodes (EPNs). In this review, we focus on the coagulation of hemolymph, which acts to stop bleeding after injury and prevent access of pathogens to the body cavity. After providing a general overview of invertebrate coagulation systems, we discuss recent findings in Drosophila melanogaster which demonstrate that clots protect against EPN infections. Detailed analysis at the cellular level provided insight into the kinetics of the secretion of Drosophila coagulation factors, including non-classical modes of secretion. Roughly, clot formation can be divided into a primary phase in which crosslinking of clot components depends on the activity of Drosophila transglutaminase and a secondary, phenoloxidase (PO)-dependent phase, characterized by further hardening and melanization of the clot matrix. These two phases appear to play distinct roles in two commonly used EPN infection models, namely Heterorhabditis bacteriophora and Steinernema carpocapsae. Finally, we discuss the implications of the coevolution between parasites such as EPNs and their hosts for the dynamics of coagulation factor evolution.
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| 15. |
- Dziedziech, Alexis, et al.
(författare)
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High-Resolution Infection Kinetics of Entomopathogenic Nematodes Entering Drosophila melanogaster
- 2020
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Ingår i: Insects. - : MDPI AG. - 2075-4450. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Entomopathogenic nematodes (EPNs) have been a useful model for studying wound healing in insects due to their natural mechanism of entering an insect host either through the cuticle or an orifice. While many experiments have shed light on nematode and host behavior, as well as the host immune response, details regarding early nematode entry and proliferative events have been limited. Using high-resolution microscopy, we provide data on the early infection kinetics of Heterorhabditis bacteriophora and its symbiotic bacteria, Photorhabdus luminescens. EPNs appendage themselves to the host and enter through the host cuticle with a drill-like mechanism while leaving their outer sheath behind. EPNs immediately release their symbiotic bacteria in the host which leads to changes in host behavior and septicemia within 6 h while EPNs travel through the host in a predictable manner, congregating in the anterior end of the host. This paper sheds light on the entry and proliferative events of EPN infection, which will further aid in our understanding of wound healing and host immune activation at a high spatiotemporal resolution.
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| 16. |
- Dziedziech, Alexis, 1991-, et al.
(författare)
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Proto-pyroptosis : An Ancestral Origin for Mammalian Inflammatory Cell Death Mechanism in Drosophila melanogaster
- 2022
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 434:4
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Tidskriftsartikel (refereegranskat)abstract
- Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. Here we provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, we unveil key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.
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| 17. |
- Dziedziech, Alexis, 1991-
(författare)
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Timing Matters : Wounding and entomopathogenic nematode infection kinetics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over time, insects have developed complex strategies to defend themselves against presenting threats. However, in the evolutionary arms race of survival, pathogens have adapted to quickly overcome the immune response mounted by the host. In this thesis, we assess how quickly entomopathogenic nematodes (EPNs) can overcome the host, Drosophila melanogaster. We then look at the clotting reaction at a hypothetical point of entry for the nematode and bring resolution to the order of protein interaction focusing on three proteins important in the anti-nematode defense. Finally, we look closer into detail at how crystal cells secrete one of those proteins, prophenoloxidase (PPOII) using a mode of programmed cell death. (Paper I) In the course of EPN infection, little was known about how quickly the worms can overcome the host immune system. Here we found that after penetrating the host, EPNs cause septicemia within 4 to 6 hours. (Paper II) Three proteins, Glutactin (Glt), Transglutaminase (Tg), and PPOII have been found to be important in the anti-nematode response. Here we created GFP-tagged fly constructs to follow their role in clot formation. In early clot formation, Tg was immediately secreted from hemocytes though it was localized around the cell membrane, Glt then entered clot fibers followed by PPOII which acted in late clot formation. (Paper III) Here we looked closer into Tg and PPOII secretion variability. PPOII from immature, but not mature crystal cells colocalized with a membrane marker. Tg, when driven with a pan tissue driver, was found located in clotting fibers, in contrast with paper II. (Paper IV) In an in vivo immune scenario, crystal cells were recruited to the wound site and burst rapidly in a caspase-dependent manner. We demonstrate that the mode of programmed cell death, pyroptosis, exists in Drosophila by way of convergent evolution.This thesis brings to light the variation found within the infection process for EPNs as well as the clotting response based on larval age, tissue type, and the maturity of a single cell type. Timing in each of these immune scenarios can give very different indications about the kind of immune response mounted and even the role of an individual cell.
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| 18. |
- Fors, Lisa, et al.
(författare)
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Differences in Cellular Immune Competence Explain Parasitoid Resistance for Two Coleopteran Species
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:9
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Tidskriftsartikel (refereegranskat)abstract
- The immune defence of an organism is evolving continuously, causing counteradaptations in interacting species, which in turn affect other ecological and evolutionary processes. Until recently comparative studies of species interactions and immunity, combining information from both ecological and immunological fields, have been rare. The cellular immune defense in insects, mainly mediated by circulating hemocytes, has been studied primarily in Lepidoptera and Diptera, whereas corresponding information about coleopteran species is still scarce. In the study presented here, we used two closely related chrysomelids, Galerucella pusilla and G. calmariensis (Coleoptera), both attacked by the same parasitoid, Asecodes parviclava (Hymenoptera). In order to investigate the structure of the immune system in Galerucella and to detect possible differences between the two species, we combined ecological studies with controlled parasitism experiments, followed by an investigation of the cell composition in the larval hemolymph. We found a striking difference in parasitism rate between the species, as well as in the level of successful immune response (i.e. encapsulation and melanisation of parasitoid eggs), with G. pusilla showing a much more potent immune defense than G. calmariensis. These differences were linked to differences in the larval cell composition, where hemocyte subsets in both naive and parasitised individuals differed significantly between the species. In particular, the hemocytes shown to be active in the encapsulation process; phagocytes, lamellocytes and granulocytes, differ between the species, indicating that the cell composition reflects the ability to defend against the parasitoid.
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| 19. |
- Fors, Lisa, et al.
(författare)
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Geographic variation and trade-offs in parasitoid virulence
- 2016
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Ingår i: Journal of Animal Ecology. - : Wiley. - 0021-8790 .- 1365-2656. ; 85:6, s. 1595-1604
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Tidskriftsartikel (refereegranskat)abstract
- 1. Host-parasitoid systems are characterized by a continuous development of new defence strategies in hosts and counter-defence mechanisms in parasitoids. This co-evolutionary arms race makes host-parasitoid systems excellent for understanding trade-offs in host use caused by evolutionary changes in host immune responses and parasitoid virulence. However, knowledge obtained from natural host-parasitoid systems on such trade-offs is still limited.2. In this study, the aim was to examine trade-offs in parasitoid virulence in Asecodes parviclava (Hymenoptera: Eulophidae) when attacking three closely related beetles: Galerucella pusilla, Galerucella calmariensis and Galerucella tenella (Coleoptera: Chrysomelidae). A second aim was to examine whether geographic variation in parasitoid infectivity or host immune response could explain differences in parasitism rate between northern and southern sites.3. More specifically, we wanted to examine whether the capacity to infect host larvae differed depending on the previous host species of the parasitoids and if such differences were connected to differences in the induction of host immune systems. This was achieved by combining controlled parasitism experiments with cytological studies of infected larvae.4. Our results reveal that parasitism success in A. parviclava differs both depending on previous and current host species, with a higher virulence when attacking larvae of the same species as the previous host. Virulence was in general high for parasitoids from G. pusilla and low for parasitoids from G. calmariensis. At the same time, G. pusilla larvae had the strongest immune response and G. calmariensis the weakest. These observations were linked to changes in the larval hemocyte composition, showing changes in cell types important for the encapsulation process in individuals infected by more or less virulent parasitoids.5. These findings suggest ongoing evolution in parasitoid virulence and host immune response, making the system a strong candidate for further studies on host race formation and speciation.
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| 20. |
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| 21. |
- Hauling, Thomas, et al.
(författare)
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A Drosophila immune response against Ras-induced overgrowth
- 2014
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Ingår i: Biology Open. - : The Company of Biologists. - 2046-6390. ; 3:4, s. 250-260
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Tidskriftsartikel (refereegranskat)abstract
- Our goal is to characterize the innate immune response against the early stage of tumor development. For this, animal models where genetic changes in specific cells and tissues can be performed in a controlled way have become increasingly important, including the fruitfly Drosophila melanogaster. Many tumor mutants in Drosophila affect the germline and, as a consequence, also the immune system itself, making it difficult to ascribe their phenotype to a specific tissue. Only during the past decade, mutations have been induced systematically in somatic cells to study the control of tumorous growth by neighboring cells and by immune cells. Here we show that upon ectopic expression of a dominant-active form of the Ras oncogene (Ras(V12)), both imaginal discs and salivary glands are affected. Particularly, the glands increase in size, express metalloproteinases and display apoptotic markers. This leads to a strong cellular response, which has many hallmarks of the granuloma-like encapsulation reaction, usually mounted by the insect against larger foreign objects. RNA sequencing of the fat body reveals a characteristic humoral immune response. In addition we also identify genes that are specifically induced upon expression of Ras(V12). As a proof-of-principle, we show that one of the induced genes (santa-maria), which encodes a scavenger receptor, modulates damage to the salivary glands. The list of genes we have identified provides a rich source for further functional characterization. Our hope is that this will lead to a better understanding of the earliest stage of innate immune responses against tumors with implications for mammalian immunity.
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| 22. |
- Hauptmann, Giselbert, et al.
(författare)
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Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos
- 2016
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :107
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Tidskriftsartikel (refereegranskat)abstract
- To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.
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| 23. |
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| 24. |
- Hyrsl, Pavel, et al.
(författare)
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Clotting Factors and Eicosanoids Protect against Nematode Infections
- 2011
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 3:1, s. 65-70
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Tidskriftsartikel (refereegranskat)abstract
- We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation.
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| 25. |
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| 26. |
- Keehnen, Naomi, et al.
(författare)
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Geographic variation in hemocyte diversity and phagocytic propensity shows a diffuse genomic signature in the green veined white butterfly
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them is likely to result in local adaptation in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed and processed for antigen presentation. We investigated the phenotypic and genetic variation related to phagocytosis, in two allopatric populations of the butterfly Pieris napi. We found that the populations differ in their hemocyte composition, and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. However, no evidence for divergence in phagocytosis-related genes was observed, though an enrichment of genes involved in glutamine metabolism was found, which have recently been linked to immune cell differentiation in mammals.
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| 27. |
- Keehnen, Naomi L.P., et al.
(författare)
-
A Population Genomic Investigation of Immune Cell Diversity and Phagocytic Capacity in a Butterfly
- 2021
-
Ingår i: Genes. - : MDPI AG. - 2073-4425. ; 12:2
-
Tidskriftsartikel (refereegranskat)abstract
- Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences.
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| 28. |
- Keehnen, Naomi L. P., 1987-, et al.
(författare)
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Insect Antimicrobial Defences : A Brief History, Recent Findings, Biases, and a Way Forward in Evolutionary Studies
- 2017
-
Ingår i: Insect Immunity. - London : Academic Press. - 9780128117750 - 9780128117767 ; , s. 1-33
-
Bokkapitel (refereegranskat)abstract
- We propose that an evolutionary and phenotype-driven approach, harnessing current technological developments, has much to offer for our understanding of insect immunity. After briefly reviewing the history of the discovery of canonical immune system, the current understanding of its components is reviewed and then we argue that the current paradigm of research may be biassed due to (a) its limited taxonomic perspective, (b) the evolutionary time scale being studied, and (c) a focus primarily if not exclusively, upon the canonical, humoural gene set. For the rest of the review, we then discuss the importance of a phenotype down approach as an understudied perspective, exemplified by the need for understanding the basis of cellular responses and wounding as a source of selection on immunity in the wild. We propose that research on those topics almost certainly will provide new insights into the evolution of the insect immune system.
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| 29. |
- Keehnen, Naomi L. P., et al.
(författare)
-
Physiological Tradeoffs of Immune Response Differs by Infection Type in Pieris napi
- 2021
-
Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- Understanding the tradeoffs that result from successful infection responses is central to understanding how life histories evolve. Gaining such insights, however, can be challenging, as they may be pathogen specific and confounded with experimental design. Here, we investigated whether infection from gram positive or negative bacteria results in different physiological tradeoffs, and whether these infections impact life history later in life (post-diapause development), in the butterfly Pieris napi. During the first 24 h after infection (3, 6, 12, and 24 h), after removing effects due to injection, larvae infected with Micrococcus luteus showed a strong suppression of all non-immunity related processes while several types of immune responses were upregulated. In contrast, this tradeoff between homeostasis and immune response was much less pronounced in Escherichia coli infections. These differences were also visible long after infection, via weight loss and slower development, as well as an increased mortality at higher infection levels during later stages of development. Individuals infected with M. luteus, compared to E. coli, had a higher mortality rate, and a lower pupal weight, developmental rate and adult weight. Further, males exhibited a more negative impact of infection than females. Thus, immune responses come at a cost even when the initial infection has been overcome, and these costs are likely to affect later life history parameters with fitness consequences.
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| 30. |
- Keehnen, Naomi L. P., et al.
(författare)
-
The consequences of surviving infection across the metamorphic boundary : tradeoff insights from RNAseq and life history measures
- 2024
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The broad diversity of insect life has been shaped, in part, by pathogen pressure, yet the influence of injury and infection during critical periods of development is understudied. During development, insects undergo metamorphosis, wherein the organism experiences a dramatic shift in their overall morphology, and physiology. In temperate zones, metamorphosis is often directly followed by a developmental arrest called diapause, for which the insect needs to acquire enough energy reserves before the onset of winter. We investigated the long-term effects of injury and infection using two bacteria in the butterfly Pieris napi, revealing that the negative consequences of bacterial infection carry across the metamorphic boundary. Initial direct effects of infection were weight loss and slower development, as well as an increased mortality at higher infection levels. The detrimental effects were stronger in the gram-positive Micrococcus luteus compared to gram-negative Escherichia coli. Transcriptome-wide differences between the two bacteria were already observed in the gene expression profile of the first 24 hours after infection. Larvae infected with M. luteus showed a strong suppression of all non-immunity related processes, with several types of immune responses being activated. The impact of these transcriptomic changes, a tradeoff between homeostasis and immune response, were visible in the life history data, wherein individuals infected with M. luteus had the highest mortality rate, along with the lowest pupal weight, developmental rate and adult weight of all the treatments. Overall, we find that the cost of infection and wounding in the final larval instar carries over the metamorphic boundary, and is expected to negatively affect their lifetime fitness.
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| 31. |
- Khalili, Dilan, et al.
(författare)
-
Anti-Fibrotic Activity of an Antimicrobial Peptide in a Drosophila Model
- 2021
-
Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 13:6, s. 376-390
-
Tidskriftsartikel (refereegranskat)abstract
- Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions.
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| 32. |
- Khalili, Dilan, et al.
(författare)
-
Chitinase-like proteins promoting tumorigenesis through disruption of cell polarity via enlarged endosomal vesicles
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Chitinase-like proteins (CLPs) are associated with tissue-remodelling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, the CLP's role in tumors is far from clear. Here, we utilize Drosophila melanogaster to investigate the function of CLPs (imaginal disc growth factors; Idgf's) in RasV12 dysplastic salivary glands. We find one of the Idgf's members, Idgf3, is transcriptionally induced tissue and cell-autonomously and in a non-canonical JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EVs) that promote tumor progression by disrupting cytoskeletal organization, independent of Rab5 and Rab11. The process is mediated via Rac1 and the downstream component, αSpectrin, localized to the EVs. Our data provide new insight into tissue-autonomous CLP function in tumors and identifies specific targets for tumor control.
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| 33. |
- Khalili, Dilan, 1988-, et al.
(författare)
-
Chitinase-like proteins promoting tumorigenesis through disruption of cell polarity via enlarged endosomal vesicles
- 2023
-
Ingår i: Frontiers in Oncology. - 2234-943X. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Chitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP’s role in tumors is far from clear.Methods: Here, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf’s) in RasV12 dysplastic salivary glands.Results and discussion: We find one of the Idgf’s members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.
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| 34. |
- Khalili, Dilan, 1988-, et al.
(författare)
-
Single-cell sequencing of tumor-associated macrophages in a Drosophila model
- 2023
-
Ingår i: Frontiers in Immunology. - 1664-3224. ; 14
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized.Methods: We use a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (RasV12) to study dysplastic growth during early tumor progression. We performed single-cell RNA-sequencing of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells.Results and discussion: We identified five distinct hemocyte clusters. In addition to RasV12 larvae, we included a tumor model where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells—they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors—antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, we provide evidence for transcript transfer between hemocytes and possibly other tissues. Taken together, our data support the usefulness of Drosophila to study the response against tumors at the organismic level.
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| 35. |
- Khalili, Dilan, 1988-
(författare)
-
Stress and immune signaling in a Drosophila tumor model
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer cells contain multiple biological alterations that allow them to escape from host surveillance mechanisms. One of the mechanisms that play an essential role in host protection against tumor growth is immunity. However, the immune system may act as a double-edged sword with the potential to both promote and limit tumor growth in a context-dependent manner. This involves both internal and external signaling events such as stress signaling pathways but also communication between cells and/or between cells and the extracellular matrix (ECM). In this thesis, Drosophila melanogaster (the fruit fly) was used to understand the role of two immune-related components, namely the antimicrobial peptide Drosomycin (Drs) and a chitinase-like protein (Idgf3), in a tumor model that involves a tubular organ, namely the salivary glands.In Paper I we investigated Drs function and regulation upon expression of the oncogene RasV12. Initially, Drs was upregulated in the whole SG upon RasV12 expression. However, at the later stage of the tumor, Drs expression was restricted to the proximal region. In contrast, at the distal region, the hallmarks of cancer phenotypes, such as activation of the pro-tumorogenic JNK pathway, adhering immune cells and production of reactive oxygen species (ROS), were elevated. By overexpressing Drs in the distal region, we found that Drs interferes with most cancer hallmarks, including the JNK-pathway, recruitment of immune cells, and ROS production.In Paper II we further characterized the hallmarks of cancer in our model system by addressing external and internal changes and whether Drs may influence them. At the extracellular compartment, we demonstrate the redistribution of the ECM in tumors, recruitment of immune components, including prophenoloxidases (PPOs) and Drs, and identified F-actin as a part of the ECM. Intracellularly, the organs' primary function, secretion, is lost, and the cell’s epithelial organization is disturbed. Drs reversed the majority of these changes.In Paper III we addressed the role of Idgf3 and its effect on external and internal cues. Initially, we found that Idgf3 was induced in the RasV12 salivary glands. Upon knock-down of Idgf3, the cellular organization was restored, and tumor growth was limited. Moreover, Idgf3 expression was correlatively increasing with the progression of the tumor. In line with Paper I, we found a similar correlation with the JNK pathway. Through genetic experiments, we show JNK-mediated regulation of Idgf3 through ROS. By addressing the subcellular localization of Idgf3, we found the protein internalized within enlarged vesicles, which were coated with a cytoskeletal protein, Spectrin. Furthermore, the formation of enlarged vesicles promoted tumor progression through loss of cellular organization. Taken together, the findings presented here emphasize the complexity of the immune system and its function in tumor progression. Further studies are necessary to understand the potential for tumor therapy.
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| 36. |
- Korayem, Ahmed, et al.
(författare)
-
Evidence for an immune function of lepidopteran silk proteins
- 2007
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 352:2, s. 317-322
-
Tidskriftsartikel (refereegranskat)abstract
- Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.
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| 37. |
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| 38. |
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| 39. |
- Krautz, Robert, et al.
(författare)
-
Damage signals in the insect immune response
- 2014
-
Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 5
-
Forskningsöversikt (refereegranskat)abstract
- Insects and mammals share an ancient innate immune system comprising both humoral and cellular responses. The insect immune system consists of the fat body, which secretes effector molecules into the hemolymph and several classes of hemocytes, which reside in the hemolymph and of protective border epithelia. Key features of wound- and immune responses are shared between insect and mammalian immune systems including the mode of activation by commonly shared microbial (non-self) patterns and the recognition of these patterns by dedicated receptors. It is unclear how metazoan parasites in insects, which lack these shared motifs, are recognized. Research in recent years has demonstrated that during entry into the insect host, many eukaryotic pathogens leave traces that alert potential hosts of the damage they have afflicted. In accordance with terminology used in the mammalian immune systems, these signals have been dubbed danger- or damage-associated signals. Damage signals are necessary byproducts generated during entering hosts either by mechanical or proteolytic damage. Here, we briefly review the current stage of knowledge on how wound closure and wound healing during mechanical damage is regulated and how damage-related signals contribute to these processes. We also discuss how sensors of proteolytic activity induce insect innate immune responses. Strikingly damage-associated signals are also released from cells that have aberrant growth, including tumor cells. These signals may induce apoptosis in the damaged cells, the recruitment of immune cells to the aberrant tissue and even activate humoral responses. Thus, this ensures the removal of aberrant cells and compensatory proliferation to replace lost tissue. Several of these pathways may have been co-opted from wound healing and developmental processes.
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| 40. |
- Krautz, Robert, 1986-
(författare)
-
Drosophila immune responses in a model for epithelial hypertrophy
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis, differentiation and proliferation have to be tightly balanced and thus regulated to maintain tissue homeostasis. Stress, metabolic cues, genetic variability, infections and physiological host-commensal interactions influence this balance and thus need to be integrated. Therefore, beyond the discrimination between self and non-self (i.e., foreign) also damage inflicted on tissues under sterile conditions is perceived by the immune system due to altered tissue integrity. Growing knowledge of the interaction between the immune system and wounded or more generally altered tissues allows inferring on anti-tumorous immune responses, too. Despite the lack of adaptive immunity, Drosophila mounts solid and versatile innate immune responses that functionally and molecularly share many properties with their vertebrate counterparts. In fact, tissue overgrowth, tissue dysplasia or endogenous danger signaling activate systemic Toll-signaling in the fat body indicating a role for the Drosophila immune system in maintaining tissue homeostasis.Here we characterize systemic and local immune responses towards altered or transformed tissues by using a Drosophila hypertrophy model, which is based on the overexpression of a dominant-active variant of the small GTPase Ras (Ras85DG12V) in salivary glands and wing discs. We characterized the strong induction of hemocyte recruitment to the glands as a consequence of JNK-dependent MMP1-expression and basal membrane degradation. Apart from this cellular immune reaction, transcriptome profiling revealed comprehensive humoral immune responses mounted by the fat body that involved signatures of Toll- and imd-activation. Moreover, a novel tissue-autonomous response that was spatially restricted to the anterior end of the RasV12-expressing salivary gland itself was identified. While multiple immune genes were found to be upregulated in the anterior compartment as detected by RNA sequencing, particular focus was given to the effector peptide Drosomycin (Drs). Overexpression of Drs with RasV12 in the entire gland similar to the inhibition of the JNK-pathway was able to selectively rescue a characteristic set of RasV12-induced phenotypes, which ultimately blocks the recruitment of hemocytes. Thereby, local immune-related responses in RasV12-expressing salivary glands are able to restrict the tissue damage induced by hypertrophic growth.
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| 41. |
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| 42. |
- Krautz, Robert, et al.
(författare)
-
Tissue-autonomous immune response regulates stress signalling during hypertrophy
- 2020
-
Ingår i: eLIFE. - 2050-084X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via Ras(V12)-overexpression in the postmitotic salivary glands of Drosophila larvae, we overrode the glands adaptability to growth signals and induced hypertrophy. The accompanied loss of tissue integrity, recognition by cellular immunity and cell death are all buffered by blocking stress signalling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing salivary glands to cope with temporary stress, continuous Drosomycin expression in Ras(V12)-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues.
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| 43. |
- Kubrak, Olga I., et al.
(författare)
-
Characterization of Reproductive Dormancy in Male Drosophila melanogaster
- 2016
-
Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Insects are known to respond to seasonal and adverse environmental changes by entering dormancy, also known as diapause. In some insect species, including Drosophila melanogaster, dormancy occurs in the adult organism and postpones reproduction. This adult dormancy has been studied in female flies where it is characterized by arrested development of ovaries, altered nutrient stores, lowered metabolism, increased stress and immune resistance and drastically extended lifespan. Male dormancy, however, has not been investigated in D. melanogaster, and its physiology is poorly known in most insects. Here we show that unmated 3-6 h old male flies placed at low temperature (11 degrees C) and short photoperiod (10 Light:14 Dark) enter a state of dormancy with arrested spermatogenesis and development of testes and male accessory glands. Over 3 weeks of diapause we see a dynamic increase in stored carbohydrates and an initial increase and then a decrease in lipids. We also note an up-regulated expression of genes involved in metabolism, stress responses and innate immunity. Interestingly, we found that male flies that entered reproductive dormancy do not attempt to mate females kept under non-diapause conditions (25 degrees C, 1 2L:1 2D), and conversely non-diapausing males do not mate females in dormancy. In summary, our study shows that male D. melanogaster can enter reproductive dormancy. However, our data suggest that dormant male flies deplete stored nutrients faster than females, studied earlier, and that males take longer to recover reproductive capacity after reintroduction to non-diapause conditions.
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| 44. |
- Kubrak, Olga I., et al.
(författare)
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The Sleeping Beauty : How Reproductive Diapause Affects Hormone Signaling, Metabolism, Immune Response and Somatic Maintenance in Drosophila melanogaster
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11
-
Tidskriftsartikel (refereegranskat)abstract
- Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagonand insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, drosomycin and cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. Our study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy.
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| 45. |
- Kucerova, Lucie, et al.
(författare)
-
Slowed aging during reproductive dormancy is reflected in genome-wide transcriptome changes in Drosophila melanogaster
- 2016
-
Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 17
-
Tidskriftsartikel (refereegranskat)abstract
- Background: In models extensively used in studies of aging and extended lifespan, such as C. elegans and Drosophila, adult senescence is regulated by gene networks that are likely to be similar to ones that underlie lifespan extension during dormancy. These include the evolutionarily conserved insulin/IGF, TOR and germ line-signaling pathways. Dormancy, also known as dauer stage in the larval worm or adult diapause in the fly, is triggered by adverse environmental conditions, and results in drastically extended lifespan with negligible senescence. It is furthermore characterized by increased stress resistance and somatic maintenance, developmental arrest and reallocated energy resources. In the fly Drosophila melanogaster adult reproductive diapause is additionally manifested in arrested ovary development, improved immune defense and altered metabolism. However, the molecular mechanisms behind this adaptive lifespan extension are not well understood. Results: A genome wide analysis of transcript changes in diapausing D. melanogaster revealed a differential regulation of more than 4600 genes. Gene ontology (GO) and KEGG pathway analysis reveal that many of these genes are part of signaling pathways that regulate metabolism, stress responses, detoxification, immunity, protein synthesis and processes during aging. More specifically, gene readouts and detailed mapping of the pathways indicate downregulation of insulin-IGF (IIS), target of rapamycin (TOR) and MAP kinase signaling, whereas Toll-dependent immune signaling, Jun-N-terminal kinase (JNK) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways are upregulated during diapause. Furthermore, we detected transcriptional regulation of a large number of genes specifically associated with aging and longevity. Conclusions: We find that many affected genes and signal pathways are shared between dormancy, aging and lifespan extension, including IIS, TOR, JAK/STAT and JNK. A substantial fraction of the genes affected by diapause have also been found to alter their expression in response to starvation and cold exposure in D. melanogaster, and the pathways overlap those reported in GO analysis of other invertebrates in dormancy or even hibernating mammals. Our study, thus, shows that D. melanogaster is a genetically tractable model for dormancy in other organisms and effects of dormancy on aging and lifespan.
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| 46. |
- Kucerova, Lucie, et al.
(författare)
-
The Drosophila Chitinase-Like Protein IDGF3 Is Involved in Protection against Nematodes and in Wound Healing
- 2016
-
Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 8:2, s. 199-210
-
Tidskriftsartikel (refereegranskat)abstract
- Chitinase-like proteins (CLPs) of the 18 glycosyl hydrolase family retain structural similarity to chitinases but lack enzymatic activity. Although CLPs are upregulated in several human disorders that affect regenerative and inflammatory processes, very little is known about their normal physiological function. We show that an insect CLP (Drosophila imaginal disc growth factor 3, IDGF3) plays an immune-protective role during entomopathogenic nematode (EPN) infections. During these infections, nematodes force their entry into the host via border tissues, thus creating wounds. Whole-genome transcriptional analysis of nematode-infected wildtype and Idgf3 mutant larvae have shown that, in addition to the regulation of genes related to immunity and wound closure, IDGF3 represses Jak/STAT and Wingless signaling. Further experiments have confirmed that IDGF3 has multiple roles in innate immunity. It serves as an essential component required for the formation of hemolymph clots that seal wounds, and Idgf3 mutants display an extended developmental delay during wound healing. Altogether, our findings indicate that vertebrate and invertebrate CLP proteins function in analogous settings and have a broad impact on inflammatory reactions and infections. This opens the way to further genetic analysis of Drosophila IDGF3 and will help to elucidate the exact molecular context of CLP function.
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| 47. |
- Kunc, Martin, et al.
(författare)
-
Monitoring the effect of pathogenic nematodes on locomotion of Drosophila larvae
- 2017
-
Ingår i: fly. - : Informa UK Limited. - 1933-6934 .- 1933-6942. ; 11:3, s. 208-217
-
Tidskriftsartikel (refereegranskat)abstract
- One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and Drosophila melanogaster larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing Drosophila strains. These differences were further influenced by the presence of nematodes. Given a choice, Drosophila larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction.
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| 48. |
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| 49. |
- Lesch, Christine, et al.
(författare)
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A role for Hemolectin in coagulation and immunity in Drosophila melanogaster
- 2007
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 31:12, s. 1255-1263
-
Tidskriftsartikel (refereegranskat)abstract
- Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacteria infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.
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| 50. |
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