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Activation of the Cellular Immune Response in Drosophila melanogaster Larvae
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Hultmark)
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors.

In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes.

In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation.

In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction.

In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1.

Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University , 2015. , s. 41
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1741
Nyckelord [en]
Drosophila melanogaster, immunity, blood cells, hematopoiesis, flow cytometry, in vivo imaging, genetic screens, Tl10b, fat body, Toll signaling
Nationell ämneskategori
Immunologi
Forskningsämne
molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-106981ISBN: 978-91-7601-317-5 (tryckt)OAI: oai:DiVA.org:umu-106981DiVA, id: diva2:845929
Disputation
2015-09-07, Major Groove, Byggnad 6L, Umeå, 12:30 (Engelska)
Opponent
Handledare
Tillgänglig från: 2015-08-17 Skapad: 2015-08-13 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
Delarbeten
1. A directed screen for genes involved in Drosophila blood cell activation
Öppna denna publikation i ny flik eller fönster >>A directed screen for genes involved in Drosophila blood cell activation
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2004 (Engelska)Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, nr 39, s. 14192-14197Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

An attack by a parasitic wasp activates a vigorous cellular immune response in Drosophila larvae. This response is manifested by an increased number of circulating cells, the hemocytes, and by the appearance of a specialized class of hemocyte, the lamellocytes, which participate in the encapsulation and killing of the parasite. To study the molecular mechanisms of this response, we have overexpressed different genes in the hemocytes, by using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-GAL4 driver. Multiple transgenes were tested, representing several important signaling pathways. We found that the proliferation response and the activation of lamellocyte formation are independent phenomena. A drastic increase in the number of circulating hemocytes is caused by receptor tyrosine kinases, such as Egfr, Pvr, and Alk, as well as by the downstream signaling components Ras85D and pointed, supporting the notion that the Ras-mitogen-activated protein kinase pathway regulates hemocyte numbers. In the case of Pvr and Alk, this phenotype also is accompanied by lamellocyte formation. By contrast, constitutively active hopscotch and hemipterous give massive activation of lamellocyte formation with little or no increase in total hemocyte numbers. This finding indicates that both the Jak/Stat and the Jun kinase pathways affect lamellocyte formation. Still other signals, mediated by aop(ACT), Toll(10b), and Rac1 expression, cause a simultaneous increase in lamellocyte and total cell numbers, and the same effect is seen when WNT signaling is suppressed. We conclude that the activation of a cellular response is complex and affected by multiple signaling pathways.

Ort, förlag, år, upplaga, sidor
Washington: National Academy of Sciences, 2004
Nyckelord
Animals, Drosophila/*genetics/*immunology/parasitology, Drosophila Proteins/genetics/metabolism, Female, Gene Expression, Hemocytes/enzymology/immunology/parasitology/*physiology, Larva/immunology/metabolism/parasitology, Male, Phenotype, Receptor Protein-Tyrosine Kinases/metabolism, Recombinant Fusion Proteins/genetics/metabolism, Signal Transduction, Transgenes/*genetics, Wasps/immunology, beta-Galactosidase/genetics, ras Proteins/metabolism
Nationell ämneskategori
Infektionsmedicin
Identifikatorer
urn:nbn:se:umu:diva-17054 (URN)10.1073/pnas.0403789101 (DOI)000224211400042 ()15381778 (PubMedID)
Tillgänglig från: 2007-10-28 Skapad: 2007-10-28 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
2. Infection-induced proliferation is a central hallmark of the activation of the cellular immune response in Drosophila larvae.
Öppna denna publikation i ny flik eller fönster >>Infection-induced proliferation is a central hallmark of the activation of the cellular immune response in Drosophila larvae.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

Blood cells have important roles in immune reactions in all metazoan species. In Drosophila melanogaster larvae, phagocytic plasmatocytes are the main blood cell (hemocyte) type. Lamellocytes participate in encapsulating foreign objects and are formed in response to parasitoid wasps laying their eggs into the hemocoel of the larvae. The immune reaction against wasps requires controlled recruitment and action of hemocytes from the lymph glands, sessile islets and circulation. However, the contribution of these different hematopoietic compartments to the immune-induced hemocyte pool remains unclear. We used eater-GFP and MSNF9MO-mCherry to fluorescently tag plasmatocytes and lamellocytes, respectively, and utilized flow cytometry and in vivo imaging to assess the hemocyte numbers and types in circulation and in sessile compartments after infection by three wasp species of the genus Leptopilina. We detected five different hemocyte types based on fluorescence, and a population of non-fluorescent cells. While non-infected larvae generally had only one, eaterGFP-high plasmatocyte population, early after wasp infection a new, eaterGFP-low cell population appeared in circulation. EaterGFP-high and –low cells both accumulated msnCherry during the immune response, and formed two cell lineages. Whereas the eaterGFP-low cells gradually lost GFP, the eaterGFP-high cells retained it at high levels. We suggest that eaterGFP-low cells represent an immune-induced hemocyte precursor cell pool, which, via a prelamellocyte stage, gives rise to lamellocytes. EaterGFP-high plasmatocytes also differentiated into large, msnCherry-positive hemocytes on wasp eggs, but these cells retain plasmatocyte identity. Importantly, all hemocyte types, except for lamellocytes, were able to divide after wasp infection, contributing to the increased hemocyte numbers after infection. We conclude that orchestrated differentiation and division of different hemocyte types in circulation and in sessile compartment is key to a successful immune response against parasitoid wasps.

Nationell ämneskategori
Immunologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-106978 (URN)
Tillgänglig från: 2015-08-13 Skapad: 2015-08-13 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
3. Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility
Öppna denna publikation i ny flik eller fönster >>Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-92811 (URN)
Tillgänglig från: 2014-09-04 Skapad: 2014-09-04 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
4. Control of Drosophila blood cell activation via toll signaling in the fat body
Öppna denna publikation i ny flik eller fönster >>Control of Drosophila blood cell activation via toll signaling in the fat body
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2014 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 8, artikel-id e102568Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. Here we have studied this response in detail, and found that Toll signaling in several different tissues can activate a cellular immune defense, and that this response does not require Toll signaling in the blood cells themselves. Like in the humoral immune response, we show that Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen.

Ort, förlag, år, upplaga, sidor
Public library of science, 2014
Nyckelord
Drosophila, toll signaling, immunity, hemocytes, fat body
Nationell ämneskategori
Cell- och molekylärbiologi Mikrobiologi
Forskningsämne
molekylärbiologi; biologi
Identifikatorer
urn:nbn:se:umu:diva-92747 (URN)10.1371/journal.pone.0102568 (DOI)000339993900003 ()25102059 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet
Tillgänglig från: 2014-09-02 Skapad: 2014-09-02 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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