Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila.

Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.

Dermatophagoides pteronyssinus major allergen 1 activates the innate immune response of the fruit fly Drosophila melanogaster.

Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.

The microbial composition of larval airways from Drosophila melanogaster differ between specimens from laboratory and natural habitats.

BACKGROUND: The fruit fly Drosophila melanogaster lives in natural habitats and has also long been used as a model organism in biological research. In this study, we used a molecular barcoding approach to analyse the airways microbiome of larvae of D. melanogaster, which were obtained from eggs of flies of the laboratory strain w1118 and from immune deficient flies (NF-kB-K), and from wild-caught flies. To assess intergenerational transmission of microbes, all eggs were incubated under the same semi-sterile conditions. RESULTS: The airway microbiome of larvae from both lab-strains was dominated by the two families Acetobacteraceae and Lactobacillaceae, while larvae from wild-caught flies were dominated by Lactobacillaceae, Anaplasmataceae and Leuconostocaceae. Barcodes linked to Anaplasmataceae could be further assigned to Wolbachia sp., which is a widespread intracellular pathogen in arthropods. For Leuconostoceae, the most abundant reads were assigned to Weissella sp. Both Wolbachia and Weissella affect the development of the insects. Finally, a relative high abundance of Serratia sp. was found in larvae from immune deficient relish-/- compared to w1118 and wild-caught fly airways. CONCLUSIONS: Our results show for the first time that larvae from D. melanogaster harbor an airway microbiome, which is of low complexity and strongly influenced by the environmental conditions and to a lesser extent by the immune status. Furthermore, our data indicate an intergenerational transmission of the microbiome as shaped by the environment.

A Drosophila asthma model - what the fly tells us about inflammatory diseases of the lung.

Asthma and COPD are the most relevant inflammatory diseases of the airways. In western countries they show a steeply increasing prevalence, making them to a severe burden for health systems around the world. Although these diseases are typically complex ones, they have an important genetic component. Genome-wide association studies have provided us with a relatively small but comprehensive list of asthma susceptibility genes that will be extended and presumably completed in the near future. To identify the role of these genes in the physiology and pathophysiology of the lung, genetically tractable model organisms are indispensable and murine models were the only ones that have been extensively used. An urgent demand for complementary models is present that provide specific advantages lacking in murine models, especially regarding speed and flexibility. Among the model organisms available, only the fruit fly Drosophila melanogaster shares a comparable organ composition and at least a lung equivalent. It has to be acknowledged that the fruit fly Drosophila has almost completely been ignored as a model organism for lung diseases, simply because it is devoid of lungs. Nevertheless, its airway system shows striking similarities with the one of mammals regarding its physiology and reaction towards pathogens, which holds the potential to function as a versatile model in asthma-related diseases.

Early-life exposure to tobacco smoke alters airway signaling pathways and later mortality in D. melanogaster.

Early life environmental influences such as exposure to cigarette smoke (CS) can disturb molecular processes of lung development and thereby increase the risk for later development of chronic respiratory diseases. Among the latter, asthma and chronic obstructive pulmonary disease (COPD) are the most common. The airway epithelium plays a key role in their disease pathophysiology but how CS exposure in early life influences airway developmental pathways and epithelial stress responses or survival is poorly understood. Using Drosophila melanogaster larvae as a model for early life, we demonstrate that CS enters the entire larval airway system, where it activates cyp18a1 which is homologues to human CYP1A1 to metabolize CS-derived polycyclic aromatic hydrocarbons and further induces heat shock protein 70. RNASeq studies of isolated airways showed that CS dysregulates pathways involved in oxidative stress response, innate immune response, xenobiotic and glutathione metabolic processes as well as developmental processes (BMP, FGF signaling) in both sexes, while other pathways were exclusive to females or males. Glutathione S-transferase genes were further validated by qPCR showing upregulation of gstD4, gstD5 and gstD8 in respiratory tracts of females, while gstD8 was downregulated and gstD5 unchanged in males. ROS levels were increased in airways after CS. Exposure to CS further resulted in higher larval mortality, lower larval-pupal transition, and hatching rates in males only as compared to air-exposed controls. Taken together, early life CS induces airway epithelial stress responses and dysregulates pathways involved in the fly's branching morphogenesis as well as in mammalian lung development. CS further affected fitness and development in a highly sex-specific manner.

Sex dependent effect of maternal e-nicotine on F1 Drosophila development and airways.

E-cigarettes are heavily advertised as healthier alternative to common tobacco cigarettes, leading more and more women to switch from regular cigarettes to ENDS (electronic nicotine delivery system) during pregnancy. While the noxious consequences of tobacco smoking during pregnancy on the offspring health are well-described, information on the long-term consequences due to maternal use of e-cigarettes do not exist so far. Therefore, we aimed to investigate how maternal e-nicotine influences offspring development from earliest life until adulthood. To this end, virgin female Drosophila melanogaster flies were exposed to nicotine vapor (8 µg nicotine) once per hour for a total of eight times. Following the last exposure, e-nicotine or sham exposed females were mated with non-exposed males. The F1-generation was then analyzed for viability, growth and airway structure. We demonstrate that maternal exposure to e-nicotine not only leads to reduced maternal fertility, but also negatively affects size and weight, as well as tracheal development of the F1-generation, lasting from embryonic stage until adulthood. These results not only underline the need for studies investigating the effects of maternal vaping on offspring health, but also propose our established model for analyzing molecular mechanisms and signaling pathways mediating these intergenerational changes.

Driver mutations in major lung cancer oncogenes can be analyzed in Drosophila models

Lung cancer remains the leading cause of cancer-associated mortality. Despite recent promising achievements, the overall prognosis remains very poor. In order to integrate the advantages of adapted, transgenic animal models with a high-throughput procedure on the one hand and compliance with the 3R principles on the other hand, we have established and evaluated appropriate Drosophila models. To achieve this goal, we ectopically expressed oncogenes representing the most important driver mutations exclusively in the airway system. These oncogenes were either the human oncogenes or the corresponding Drosophila orthologs. We concentrated on two complementary read-out systems, 1) early larval lethality and 2) quantification of concurrently expressed GFP as a proxy for tumor mass. We could show that ectopic expression of EgfrCA, RasV12, Raf, Rolled (MAPK), PI3K92E, Alk, Akt and Arm can induce early lethality. Thus, they can be used in a straight-forward high-throughput screening approach and can replace mouse models to a considerable extent. Moreover, we could also show that measurement of tumor mass by a concurrently expressed marker (GFP) can be used to detect positive treatment results. Our results show that our Drosophila system provides a superb in vivo inver­tebrate screening system amenable to high-throughput approaches and thus effectively complements the toolbox for the development of novel anti-lung cancer treatments, while complying with the 3R principles.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

An EGFR-Induced Drosophila Lung Tumor Model Identifies Alternative Combination Treatments.

Lung cancer is the leading cause of cancer-associated mortality. Mutations in the EGFR gene are among the most important inducers of lung tumor development, but success of personalized therapies is still limited because of toxicity or developing resistances. We expressed constitutively active EGFR (EGFRCA) exclusively in the airway system of Drosophila melanogaster and performed comprehensive phenotyping. Ectopic expression of EGFRCA induced massive hyper- and metaplasia, leading to early death. We used the lethal phenotype as a readout and screened a library of FDA-approved compounds and found that among the 1,000 compounds, only the tyrosine kinase inhibitors (TKI) afatinib, gefitinib, and ibrutinib rescued lethality in a whole-animal screening approach. Furthermore, we screened the library in the presence of a subtherapeutic afatinib dose and identified bazedoxifene as a synergistically acting compound that rescues EGFR-induced lethality. Our findings highlight the potential of Drosophila-based whole-animal screening approaches not only to identify specific EGFR inhibitors but also to discover compounds that act synergistically with known TKIs. Moreover, we showed that targeting the EGFR together with STAT-signaling is a promising strategy for lung tumor treatment.

A Drosophila model of cigarette smoke induced COPD identifies Nrf2 signaling as an expedient target for intervention.

Chronic obstructive pulmonary disease (COPD) is among the most important causes of death. Signaling systems that are relevant for tissue repair and detoxification of reactive oxygen species or xenobiotics are thought to be impaired in lungs of patients suffering from this disease. Here, we developed a simple cigarette smoke induced Drosophila model of COPD based on chronic cigarette smoke exposure that recapitulates major pathological hallmarks of the disease and thus can be used to investigate new therapeutic strategies. Chronic cigarette smoke exposure led to premature death of the animals and induced a set of phenotypes reminiscent of those seen in COPD patients, including reduced physical activity, reduced body fat, increased metabolic rate and a substantial reduction of the respiratory surface. A detailed transcriptomic analysis revealed that especially the TGF-ß, Nrf2 and the JAK/STAT signaling pathways are altered by chronic cigarette smoke exposure. Based on these results, we focused on Nrf2 signaling. A pharmacological intervention study performed with oltipraz, an activator of Nrf2 signaling, increased survival of cigarette smoke exposed animals significantly. Thus, the Drosophila COPD model recapitulates many major hallmarks of COPD and it is highly useful to evaluate the potential of alternative therapeutic strategies.

Temporal expression and localization patterns of variant surface antigens in clinical Plasmodium falciparum isolates during erythrocyte schizogony.

Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite Plasmodium falciparum possesses a number of multi-copy gene families, including var, rif, stevor and pfmc-2tm, which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on in vitro analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by P. falciparum in the human host. To investigate the expression of the var, rif-A, rif-B, stevor and pfmc-2tm family genes under conditions that mimic more closely the natural course of infection, ex vivo clinical P. falciparum isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle in vitro were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families rif-A, stevor and pfmc-2tm at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the pfmc-2tm family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and PfMC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of P. falciparum multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.