Immunity in Drosophila melanogaster--from microbial recognition to whole-organism physiology

Abstract

Since the discovery of antimicrobial peptide responses 40 years ago, the fruit fly Drosophila melanogaster has proven to be a powerful model for the study of innate immunity. Early work focused on innate immune mechanisms of microbial recognition and subsequent nuclear factor-κB signal transduction. More recently, D. melanogaster has been used to understand how the immune response is regulated and coordinated at the level of the whole organism. For example, researchers have used this model in studies investigating interactions between the microbiota and the immune system at barrier epithelial surfaces that ensure proper nutritional and immune homeostasis both locally and systemically. In addition, studies in D. melanogaster have been pivotal in uncovering how the immune response is regulated by both endocrine and metabolic signalling systems, and how the immune response modifies these systems as part of a homeostatic circuit. In this Review, we briefly summarize microbial recognition and antiviral immunity in D. melanogaster, and we highlight recent studies that have explored the effects of organism-wide regulation of the immune response and, conversely, the effects of the immune response on organism physiology.

Figure 1. Innate immunity in Drosophila melanogaster.

The organ systems of Drosophila melanogaster are analogous to those in vertebrates; the gut absorbs nutrients, whereas the fat body stores nutrients and functions as a nutrient sensor, similar to the mammalian liver and adipose tissue. The Malpighian tubules in flies carry out the same basic functions as the kidneys in vertebrates. The D. melanogaster heart is essential for the circulation of nutrients and immune cells; however, flies have an open circulatory system, rather than a vasculature, and oxygen is delivered by an independent tracheal system. Several D. melanogaster organ systems contribute to innate immune defence. Similar to mammals, who have various blood cell types, flies have several types of circulating cells, collectively known as haemocytes. Immediately upon infection, macrophage‑like plasmatocytes begin to phagocytose microbial invaders. Other circulating cells, such as the crystal cells in larvae, activate melanization, which generates bactericidal reactive oxygen species (ROS) at infection sites and promotes coagulation. Large pathogens are encapsulated by large haemocytes known as lamellocytes. The hallmark of the D. melanogaster humoral response is the inducible synthesis and secretion of antimicrobial peptides (AMPs), which are released into the haemolymph as a systemic response. The fat body is a primary systemic source of AMPs. Barrier epithelial cells in D. melanogaster are also capable of generating AMPs, similar to mammals. The trachea, Malpighian tubules and gut produce tissue‑specific AMPs in response to local microbial infection. In the gut, the inducible generation of ROS by NADPH oxidases — such as dual oxidase (Duox) and NADPH oxidase (Nox) — has a role in both pathogen infection and regulation of the gut microbiota. The D. melanogaster central nervous system coordinates both organism physiology and immunity through the secretion of hormones.

Figure 2. Immune recognition of microbial agents in Drosophila melanogaster.

Two classical signalling pathways control inducible immune responses to bacteria and fungi in D. melanogaster: the Toll pathway and the immune deficiency (Imd) pathway. The Toll pathway is active in the fat body and, together with the Imd pathway, controls the systemic production of antimicrobial peptides (AMPs). The Imd pathway is also active in barrier epithelial surfaces including the gut, and functions in antimicrobial responses together with reactive oxygen species (ROS)‑generating enzymes, such as dual oxidase (Duox). These pathways are activated in response to the detection of microbial cell wall components. Peptidoglycan recognition protein LC (PGRP‑LC) and PGRP‑LE recognize the diaminopimelic acid (DAP)‑type peptidoglycan from Gram‑negative bacteria and certain Gram‑positive bacteria, and activate the Imd pathway. PGRP‑SA and Gram‑negative bacteria‑binding protein 1 (GNBP1) recognize the lysine‑type peptidoglycan of Gram‑positive bacteria, and GNBP3 recognizes the β‑glucans of yeasts and fungi to activate Toll signalling. In addition, the Toll pathway can be activated through the sensing of danger signals — including microbial proteases — or abnormal cell death, triggering the maturation of the protease Persephone. In all variations of the Toll pathway, immune recognition activates a proteolytic cascade that culminates in the maturation of the cytokine Spätzle, which is mediated by the protease Spätzle‑processing enzyme (SPE). Toll activation ultimately leads to the nuclear translocation of the nuclear factor‑κB (NF‑κB) transcription factor Dif, to induce the expression of AMP genes such as Drosomycin, as well as other target genes. Activation of the Imd pathway leads to the nuclear translocation of the NF‑κB transcription factor Relish to activate the expression of AMP genes such as Diptericin. ROS also have important roles in antimicrobial defence and Duox activity is triggered by the recognition of uracil, which is a pathogen‑derived small molecule that activates an unidentified G protein‑coupled receptor (GPCR) and promotes the release of calcium from the endoplasmic reticulum (ER). In addition, both the Imd pathway and GPCR signalling, through a phospholipase Cβ (PLCβ)‑dependent pathway, lead to the activation of a Mekk1–p38 mitogen‑activated protein kinase (p38 MAPK) axis that promotes the sustained expression of Duox upon infection. Atf2, activating transcription factor 2; Dredd, death‑related ced‑3/Nedd2‑ like caspase; Fadd, FAS‑associated death domain orthologue; Gαq, G protein αq‑subunit; IKK, inhibitor of NF‑κB kinase (also known as Ird5); MKK3, MAPK kinase 3 (also known as Licorne); modSP, modular serine protease; Tak1, TGFβ‑activated kinase 1.

Figure 3. Nucleic acid recognition and antiviral defences in Drosophila melanogaster.

RNA viruses often encode structured RNAs or produce double‑stranded RNA (dsRNA) intermediates. Some DNA viruses produce convergent transcripts that form dsRNAs and also transcribe structured RNAs. These RNAs are recognized and cleaved by Dicer‑2 to form virus‑derived small interfering RNAs (siRNAs), which are loaded into an Argonaute 2‑containing RNA‑induced silencing complex (RISC) to silence viral expression. Dicer‑2 also initiates the transcription of antiviral genes through an as‑yet‑unidentified pathway. Endogenous retrotransposon‑encoded reverse transcriptase can generate viral‑derived cDNA, which can be further transcribed and amplify the RNA interference (RNAi) response. Another RNAi pathway, the PIWI‑interacting RNA (piRNA) pathway, protects the cell from endogenous mobile genetic elements, especially those in the germ line. In addition to these nucleic‑acid‑triggered responses, some viruses can be directly sensed by Toll‑7 to induce antiviral autophagy dependent on the conserved AKT pathway involving phosphoinositide 3‑kinase (PI3K) and target of rapamycin (Tor).

Figure 4. Drosophila melanogaster intestinal immune response to infection.

The digestive tract of Drosophila melanogaster is highly regionalized, and is composed of the ectodermal foregut, the endodermal midgut and the ectodermal hindgut. The midgut is the primary site of digestion and absorption, and is protected by a chitinous membrane known as the peritrophic matrix. The midgut is subdivided into domains that have distinct physiological properties. The gut immune response is highly regionalized; the Toll pathway is active only in the foregut and hindgut, whereas the immune deficiency (Imd) pathway mostly regulates the immune response in the midgut. In response to Imd activation, different regions of the gut express different antimicrobial peptides (AMPs); this regional regulation depends on local, developmentally programmed cues (such as local expression of the transcription factor Caudal). The recognition of extracellular and intracellular bacterial components takes place in different gut regions; immune recognition in the midgut occurs by either membrane‑bound peptidoglycan recognition protein LC (PGRP‑LC) in the anterior midgut or by intracellular PGRP‑LE in the middle and posterior midgut. The gut epithelium also produces reactive oxygen species (ROS) in response to both the natural microbiota and more virulent microorganisms by activating the NADPH oxidases dual oxidase (Duox) and Nox. Together with toxins, such as pore‑forming toxins (PFTs), excessive gut immune responses can cause tissue damage and enterocyte delamination, which is rapidly repaired through the proliferation and differentiation of intestinal stem cells. To decrease damage and maintain tissue homeostasis, negative regulators of the Imd pathway, such as poor Imd response upon knock‑in (Pirk) and the amidases PGRP‑LB and PGRP‑SC, prevent excessive activation of the immune response. Sensing of viral infection leads to the induction of antiviral extracellular signal‑regulated kinase (ERK) activation via a RAS mitogen‑activated protein kinase (MAPK) pathway in the gut epithelium.

Figure 5. Systemic regulation of Drosophila melanogaster immune responses.

The Drosophila melanogaster immune response is integrated with multiple inter‑organ regulatory circuits. a | The fat body is a central regulator of metabolic control. Here, Toll signalling through the nuclear factor‑κB (NF‑κB) transcription factor Dif inhibits insulin signalling and retards growth, whereas forkhead box subgroup O (Foxo) — the downstream target of the insulin pathway — regulates the production of antimicrobial peptides (AMPs) and anabolic genes. b | In larvae, the steroid hormone Ecdysone is produced in a secretory organ associated with the brain (known as the ring gland), whereas the source of Ecdysone is less well characterized in adult flies. Ecdysone is a major regulator of the D. melanogaster lifecycle, as well as the immune response, through controlling the expression of peptidoglycan recognition protein LC (PGRP‑LC) in the fat body and the phagocytic activity of pupal haemocytes. c | The gut microbiota further influences the insulin signalling pathway by regulating the production of Drosophila insulin‑like peptides (Dilps) in the brain. Dilp production and robust growth are triggered by acetic acid that is produced by Acetobacter species in the gut, and potentially triggered by optimization of amino acid levels provided by other components of microbiota. d | The central nervous system further regulates immune responses in the Malpighian tubules through the production of neuropeptides that are thought to trigger a nitric oxide (NO)‑dependent NF‑κB response in this organ. The faded part of this panel indicates a hypothetical role for the Imd pathway. cGMP, cyclic guanosine monophosphate; EcR, Ecdysone receptor; GC, guanylyl cyclase; Imd, immune deficiency; IPC, insulin‑producing cell; Nos, nitric oxide synthase; Tor, target of rapamycin.