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Drosophila immune responses in a model for epithelial hypertrophy
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Group Theopold)
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: urn:nbn:se:su:diva-128916ISBN: 978-91-7649-400-4OAI: oai:DiVA.org:su-128916DiVA: diva2:917747
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
List of papers
1. Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State
Open this publication in new window or tab >>Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State
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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.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-121511 (URN)10.1371/journal.pone.0136593 (DOI)000360435500023 ()
Available from: 2015-10-09 Created: 2015-10-05 Last updated: 2016-04-08Bibliographically approved
2. A Drosophila immune response against Ras-induced overgrowth
Open this publication in new window or tab >>A Drosophila immune response against Ras-induced overgrowth
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2014 (English)In: Biology Open, ISSN 2046-6390, Vol. 3, no 4, 250-260 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Innate immunity, Tumor, Oncogene, Insect immunity, Hemocytes, Encapsulation
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-113980 (URN)10.1242/bio.20146494 (DOI)000348066800003 ()
Note

AuthorCount:6;

Available from: 2015-02-18 Created: 2015-02-16 Last updated: 2016-04-08Bibliographically approved
3. Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos
Open this publication in new window or tab >>Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos
2016 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 107, e53830Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Developmental Biology, Issue 107, Digoxigenin, biotin, fluorescein, azo dye, Fast Blue, Fast Red, INT, alkaline phosphatase substrate, WISH, fruit fly
National Category
Developmental Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-128914 (URN)10.3791/53830 (DOI)
Available from: 2016-04-07 Created: 2016-04-07 Last updated: 2016-04-08Bibliographically approved
4. An innate immune response against dysplasia in a secretory organ
Open this publication in new window or tab >>An innate immune response against dysplasia in a secretory organ
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-128749 (URN)
Available from: 2016-04-03 Created: 2016-04-03 Last updated: 2016-04-08Bibliographically approved
5. Drosophila larval fat body preparations to reveal regionalized gene expression​
Open this publication in new window or tab >>Drosophila larval fat body preparations to reveal regionalized gene expression​
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-128748 (URN)
Available from: 2016-04-03 Created: 2016-04-03 Last updated: 2016-04-08Bibliographically approved

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