Molecular architecture of the fruit fly's airway epithelial immune system

Abstract

Background: Airway epithelial cells not only constitute a physical barrier, but also the first line of defence against airborne pathogens. At the same time, they are constantly exposed to reactive oxygen species. Therefore, airway epithelia cells have to possess a sophisticated innate immune system and a molecular armamentarium to detoxify reactive oxygen species. It has become apparent that deregulation of epithelial innate immunity is a major reason for the development of chronic inflammatory lung diseases. To elucidate the molecular architecture of the innate immune system of airway epithelial cells, we choose the fruit fly Drosophila melanogaster as a model, because it has the simplest type of airways, consisting of epithelial cells only. Elucidating the structure of the innate immune system of this "airway epithelial cell culture" might enable us to understand why deregulatory processes in innate immune signalling cascades lead to long lasting inflammatory events.

Results: All airway epithelial cells of the fruit fly are able to launch an immune response. They contain only one functional signal transduction pathway that converges onto NF-kappaB factors, namely the IMD-pathway, which is homologous to the TNF-alpha receptor pathway. Although vital parts of the Toll-pathway are missing, dorsal and dif, the NF-kappaB factors dedicated to this signalling system, are present. Other pathways involved in immune regulation, such as the JNK- and the JAK/STAT-pathway, are completely functional in these cells. In addition, most peptidoglycan recognition proteins, representing the almost complete collection of pattern recognition receptors, are part of the epithelial cells equipment. Potential effector molecules are different antimicrobial peptides and lysozymes, but also transferrin that can inhibit bacterial growth through iron-depletion. Reactive oxygen species can be inactivated through the almost complete armamentarium of enzymatic antioxidants that has the fly to its disposal.

Conclusion: The innate immune system of the fly's airway epithelium has a very peculiar organization. A great variety of pattern recognition receptors as well as of potential effector molecules are conspicuous, whereas signalling presumably occurs through a single NF-kappaB activating pathway. This architecture will allow reacting if confronted with different bacterial or fungal elicitors by activation of a multitude of effectors.

Figure 1

Organization of the fruit fly's airway epithelium. The airway system of the Drosophila larvae is made of simple tubes in a hierarchical order (D, modified after [31]). In all airways, starting from the primary (A) over secondary (B) up to terminal branches (C), a single layer of epithelial cells wraps around the central air-filled tube. If confronted with bacteria (Erwinia carotovora or Pseudomonas aeruginosa), the airway epithelium reacts with the expression of antimicrobial peptides (visualized using a drosomycin::gfp reporter, E). All cells, even the most terminal structures, are able to mount an immune response (arrow, F).

Figure 2

Pattern recognition receptors of the airway immune system. To identify the genes coding for pattern recognition receptors in airway epithelial cells, we performed RT-PCR experiments with RNA derived from thoroughly isolated epithelial cells. Regarding the Toll-receptors, only 4 out of 9 are expressed in these cells (A, D). All except 2 peptidoglycan recognition proteins (PGRPs) are present in the airways (B, D). From the gram-negative binding proteins, GNBP1 and 3 are present (C, D). Positive controls were performed with fatbody and blood cell derived RNA, negative control without template.

Figure 3

Signal transduction pathways in the airway epithelia. Among the 4 signal transduction pathways relevant for the innate immune response of the fly, only the Toll-pathway is not represented by all vital members (A, E). In opposite, the other pathway that terminates into activation of NF-kB factors, the IMD-pathway, is functional, because all corresponding genes are expressed (B, E). The JNK-pathway should also be functional because all relevant members are present (C). JAK/STAT signalling depends only on a very limited number of genes. The complete set of genes required for its activation is expressed in the airways (D). In addition, JAK/STAT-signalling can be visualized in the terminal tracheal structures using a STAT::gfp reporter system (F, 19).

Figure 4

Transcription factors relevant for the immune response. RT-PCR analysis of transcription factors relevant for immune responses. All 3 NF-kB factors are present in the airway epithelium. In addition, 3 out of 5 GATA-factors were found in these cells.

Figure 5

Infection with P. aeruginosa provokes increased expression of target genes of the imd-, JNK- and JAK/STAT-pathways. Semiquantitative RT-PCR analysis of target genes for the corresponding immune relevant pathway was performed with control material (c) and material from infected larvae (i). Comparison with the house keeping control gene rpl32 reveals that all candidate genes (dipt = diptericin, punch, tet42e = tetraspanin 42e, totM = turandot M vvl = ventral vein lacking) are expressed at higher levels in infected trachea.

Figure 6

Schematic delineation of antioxidant enzymes in the airway epithelial cell. A number of different enzymes that have the ability to detoxify reactive oxygen as well as reactive nitrogen species (ROS, NOS) are present in airway epithelial cells. Especially the complete SOD family and a total of four peroxiredoxins might serve as the first line of defence against oxygen radicals. On the other hand, a sophisticated glutathione based system allows detoxification of various different compounds. The dual oxidase Duox may be used to produce ROS in response to an encounter with bacterial pathogens.

Figure 7

Occurrence of functional annotation groups in the complete airway epithelial transcriptome and within the group of genes with preferential expression in the airway epithelium. Functional annotation of both sets of genes was performed using the Fatigo+ program package (23). Exemplary, some GO annotations analysis of the molecular function level 3 (structural constituent of ribosome, oxidoreductase, hydrolase, ion binding) and 4 (RNA binding, polysaccharide binding, peptidase, metal ion binding) are listed. All groups show a statistically significant (p < 0.05) difference in their occurrences between the two sets of genes.