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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
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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Number of Authors: 6
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, e0136593Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 10, no 8, e0136593
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-121511DOI: 10.1371/journal.pone.0136593ISI: 000360435500023OAI: oai:DiVA.org:su-121511DiVA: diva2:860009
Available from: 2015-10-09 Created: 2015-10-05 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Drosophila immune responses in a model for epithelial hypertrophy
Open this publication in new window or tab >>Drosophila immune responses in a model for epithelial hypertrophy
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2016. 63 p.
National Category
Immunology Genetics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-128916 (URN)978-91-7649-400-4 (ISBN)
Public defence
2016-06-01, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2016-05-09 Created: 2016-04-07 Last updated: 2016-08-17Bibliographically approved
2. Molecular characterization of the Drosophila responses towards nematodes
Open this publication in new window or tab >>Molecular characterization of the Drosophila responses towards nematodes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2016. 65 p.
Keyword
Drosophila, nematodes, infection, danger situations, immune response, coagulation, apoptosis, nitric oxide, leukemia
National Category
Biochemistry and Molecular Biology Genetics Immunology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-134346 (URN)978-91-7649-535-3 (ISBN)978-91-7649-536-0 (ISBN)
Public defence
2016-11-24, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.

Available from: 2016-11-01 Created: 2016-10-05 Last updated: 2017-05-04Bibliographically approved

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Arefin, BadrulKucerova, LucieKrautz, RobertParvin, FarjanaTheopold, Ulrich
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