Drosophila Cytokine Research Articles

Astrocyte expression of the Drosophila TNF-alpha homologue, Eiger, regulates sleep in flies.

Sleep contributes to cognitive functioning and is sufficient to alter brain morphology and function. However, mechanisms underlying sleep regulation remain poorly understood. In mammals, tumor necrosis factor-alpha (TNFα) is known to regulate sleep, and cytokine expression may represent an evolutionarily ancient mechanism in sleep regulation. Here we show that the Drosophila TNFα homologue, Eiger, mediates sleep in flies. We show that knockdown of Eiger in astrocytes, but not in neurons, significantly reduces sleep duration, and total loss-of-function reduces the homeostatic response to sleep loss. In addition, we show that neuronal, but not astrocyte, expression of the TNFα receptor superfamily member, Wengen, is necessary for sleep deprivation-induced homeostatic response and for mediating increases in sleep in response to human TNFα. These data identify a novel astrocyte-to-neuron signaling mechanism in the regulation of sleep homeostasis and show that the Drosophila cytokine, Eiger, represents an evolutionarily conserved mechanism of sleep regulation across phylogeny.

The Drosophila cytokine, GBP: A model that illuminates the yin-yang of inflammation and longevity in humans?

Our laboratories have determined that the Drosophila cytokine, Growth-blocking peptide (GBP), mediates its biological effects through the Mthl10 G-protein coupled receptor. In this Cytokine Stimulus, we discuss the functional plasticity of the GBP/Mthl10 axis, and we propose that conserved components of this regulatory network may be relevant to human health.

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP.

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. Here, we use insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. We demonstrate that a Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, we describe mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.

Drosophila Cytokine Unpaired 2 Regulates Physiological Homeostasis by Remotely Controlling Insulin Secretion

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin.

Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture

Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10 ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2 x 10(8), 1.1 x 10(9) and 3.5 x 10(10) at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2mg at day 18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus a flexible and powerful tool for manufacturing recombinant proteins.

Feedback Inhibition of JAK/STAT Signaling by Apontic Is Required to Limit an Invasive Cell Population

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. Here we report the identification of a mutant, apontic (apt), in which the migratory population expanded and separation from the epithelium was impeded. This phenotype resembled gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimicked loss of function of STAT and its key downstream target, SLBO. APT expression was induced by STAT, which bound directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes.

Psidin Is Required in Drosophila Blood Cells for Both Phagocytic Degradation and Immune Activation of the Fat Body

Phagocytic blood cells are critical to innate immune defense: They internalize and destroy microbial invaders and produce signals that trigger other immune responses. Despite this central role, the in vivo contributions of phagocytosis to systemic immune activation are not well understood. Drosophila has proven a fruitful model for the investigation of evolutionarily conserved innate immune mechanisms, including NF-kappaB-dependent transcriptional induction, RNAi in antiviral responses, and phagocytosis. The phagocytes of Drosophila encounter bacterial invaders early in infection and contribute to survival of infection. Phagocytosis in flies and mammals is highly homologous: Both rely on scavenger receptors, opsonins, and actin rearrangements for engulfment; have phagosomal cysteine proteases active at low pH; and can be subverted by similar intracellular pathogens. Although the role of Drosophila phagocytes in the activation of other immune tissues has not been clear, we show that induction of the antibacterial-peptide gene Defensin in the fat body during infection requires blood-cell contributions. We identify a gene, psidin, that encodes a lysosomal protein required in the blood cells for both degradation of engulfed bacteria and activation of fat-body Defensin. These data establish a role for the phagocytic blood cells of Drosophila in detection of infection and activation of the humoral immune response.

A Toll Receptor and a Cytokine, Toll5A and Spz1C, Are Involved in Toll Antifungal Immune Signaling in the Mosquito Aedes aegypti

The fungal-specific immune response in the mosquito Aedes aegypti involves the Toll immune pathway transduced through REL1, a homologue of the NF-kappaB transcription factor Drosophila Dorsal. The Toll receptor and its ligand, Spätzle (Spz), link extracellular immune signals to the Toll intracellular transduction pathway. Five homologues to the Drosophila Toll (Toll1) receptor (Toll1A, Toll1B, Toll5A, Toll5B, and Toll4) and three homologues to the Drosophila cytokine Spätzle (Spz1A, 1B, and 1C) were identified from genomic and cDNA sequence data bases. Toll1A, Toll5A, Toll5B, and Spz1A were specifically induced in the mosquito fat body following fungal challenge. This transcriptional up-regulation was mediated by REL1. Spz1C was constitutively expressed in the mosquito fat body, whereas Spz1B and Toll4 were primarily expressed in ovarian tissues of female mosquitoes. The transcripts of Toll1B were only detected in early stages of mosquito embryos. RNA interference knock down of Toll5A and Spz1C resulted in two phenotypes of Aedes Toll/REL1 pathway deficiency: decreased induction of Aedes Serpin-27A following fungal challenge and increased susceptibility to the entomopathogenic fungus Beauveria bassiana. These data suggest that Toll5A and Spz1C function as cytokine receptor systems specific to the Toll receptor-mediated immune response following fungal challenge in the mosquito fat body.

A Spätzle-Processing Enzyme Required for Toll Signaling Activation in Drosophila Innate Immunity

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Spätzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling.

The extracellular protein Spätzle is required for activation of the Toll signaling pathway in the embryonic development and innate immune defense of Drosophila. Spätzle is synthesized as a pro-protein and is processed to a functional form by a serine protease. We show here that the mature form of Spätzle triggers a Toll-dependent immune response after injection into the hemolymph of flies. Spätzle specifically bound to Drosophila cells and to Cos-7 cells expressing Toll. Furthermore, in vitro experiments showed that the mature form of Spätzle bound to the Toll ectodomain with high affinity and with a stoichiometry of one Spätzle dimer to two receptors. The Spätzle pro-protein was inactive in all these assays, indicating that the pro-domain sequence, which is natively unstructured, acts to prevent interaction of the cytokine and its receptor Toll. These results show that, in contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein ligand for activation and signaling.