Curly Encodes Dual Oxidase,Which Acts with Heme Peroxidase Curly Su to Shape the Adult Drosophila Wing

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. Here we show that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), we demonstrate that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, we identify a novel heme peroxidase, Curly Su (Cysu) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. Our work adds to a growing number of studies suggesting that Duox’s primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells

Mucus hypersecretion is a major pathophysiologic feature in chronic inflammatory airway diseases. Oxidative stress plays a pivotal role in this process. Recent studies have found that heparin has antioxidant effects which can reduce free radical damage. Here, we hypothesized that heparin has some influence on the expression of mucin 5AC (MUC5AC) induced by phorbol myristate acetate (PMA) in a bronchial epithelial cell line (HBE16), also we have investigated the potential mechanism involved in the process. We found that ROS, the mRNA of Duox1, EGFR and MUC5AC, as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC in the PMA group were significantly increased when compared with the control group (all P < 0.01). After pretreatment with heparin however, there was a significant decrease in ROS levels, the mRNA of Duox1, EGFR, and MUC5AC, and the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with the PMA group (all P < 0.01). MUC5AC protein in the supernatant was inhibited in a dose-dependent manner by heparin. Pretreatment with DMTU resulted in a significant decrease in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC when compared with the PMA group (all P < 0.01). When cells were pretreated with both heparin and DMTU, there was a further reduction in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with either the PMA group, heparin group, or DMTU group (all P < 0.01). Our results show that PMA can induce MUC5AC expression by activation of the Duox1-ROS-TACE-TGF-α-EGFR signaling pathway. Heparin can decrease the level of Duox1, ROS production and block the PMA-induced activation of EGFR, thus inhibiting the overexpression of mucin MUC5AC in a dose-dependent manner. In addition to reducing ROS production, heparin may also inhibit the expression of MUC5AC through other signal mechanisms.     

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.     

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis.     

Stimulation by P2X7 receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands

Background

Agonists of P2X₇ receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Δψ_m) in exocrine glands.

Methods

The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Δψ_m was estimated with tetramethylrhodamine.

Results

Activation of P2X₇ receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Δψ_m by ATP.

Conclusions

We conclude that, in rat submandibular glands, P2X₇ receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists.

General significance

Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.     

Reactive oxygen species regulate Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-alpha signaling pathways in human airway epithelial cells

Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium P. aeruginosa, an important human respiratory pathogen causing cystic fibrosis, utilizes reactive oxygen species (ROS) to up-regulate MUC5AC mucin expression. Pseudomonas aeruginosa lipopolysaccharide (PA-LPS) induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-α (TGF-α), which in turn, leads to up-regulate MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with P. aeruginosa infections.     

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

In the thyroid gland Duox2-derived H₂O₂ is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2^thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H₂O₂, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.     

Regulation of Nox and Duox enzymatic activity and expression

In recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide, and other metabolites) are produced in biological systems. Rather than being simply a by-product of aerobic metabolism, it is now recognized that specific enzymes--the Nox (NADPH oxidase) and Duox (Dual oxidase) enzymes--seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis, and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

The use of model systems to study biological functions of Nox/Duox enzymes

ROS (reactive oxygen species; including superoxide and H202) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H202 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches.     

Detecting,Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.     

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N⁶-methyladenosine (m⁶A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m⁶A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m⁶A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m⁶A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.     

The cannabinoid WIN55,212-2 protects against oxidized LDL-induced inflammatory response in murine macrophages

The endocannabinoid system has recently been attracted interest for its anti-inflammatory and anti-oxidative properties. In this study, we investigated the role of the endocannabinoid system in regulating the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in macrophages. RAW264.7 mouse macrophages and peritoneal macrophages isolated from Sprague-Dawley (SD) rats were exposed to oxLDL with or without the synthetic cannabinoid WIN55,212-2. To assess the inflammatory response, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF- α) levels were determined, and activation of the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B signaling pathways were assessed. We observed that: i) oxLDL strongly induced ROS generation and TNF- α secretion in murine macrophages; ii) oxLDL-induced TNF- α and ROS levels could be lowered considerably by WIN55,212-2 via inhibition of MAPK (ERK1/2) signaling and NF-kappa B activity; and iii) the effects of WIN55212-2 were attenuated by the selective CB2 receptor antagonist AM630. These results demonstrate the involvement of the endocannabinoid system in regulating the oxLDL-induced inflammatory response in macrophages, and indicate that the CB2 receptor may offer a novel pharmaceutical target for treating atherosclerosis.     

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes

Background  

NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.     

Several MAMPs,including chitin fragments,enhance AtPep-triggered oxidative burst independently of wounding

AtPeps are a family of small peptides in Arabidopsis that are believed to act as endogenous amplifiers of the plant’s innate immune response. In our recent study,¹⁰ we showed that in Arabidopsis leaf disks, bacterial MAMPs (microbe-associated molecular patterns) such as the flagellin derived elicitor flg22, greatly enhanced the release of reactive oxygen species (ROS) triggered by a subsequent AtPep-perception. This enhanced ROS production could be a hallmark either of improved local defense or of the initiation of ROS-based systemic signaling. Here, we established a superior ROS detection system based on a new derivative of luminol (L-012). With this sensitive system we were able to show that chitin, too, acts as an enhancer of AtPep-triggered ROS, linking this specific defense response amplification also to the recognition of fungal pathogens. In addition we used the more sensitive ROS assay to transfer the experimental setup from cut leaf disks to unwounded seedlings. Thereby we revealed that wounding is not a prerequisite to enable the MAMP-induced enhancement of the AtPep-triggered ROS response.     

Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.