InteractiveFly: GeneBrief

NADPH oxidase: Biological Overview | References


Gene name - NADPH oxidase

Synonyms -

Cytological map position - 53B3-53B4

Function - enzyme

Keywords - generation of reactive oxygen species, Nox knockdown in mature follicle cells leads to a reduction in superoxide and to defective ovulation, Nox in required for induction of the secreted Turandot family proteins through the action of Unpaired proteins, extracellular actin detection via Nox dependent Src-family kinase-dependent cascade is means of detecting cell injury - immune response - expressed in midgut

Symbol - Nox

FlyBase ID: FBgn0085428

Genetic map position - chr3R:17,750,129-17,762,481

NCBI classification - Calcium ion binding; superoxide-generating NADPH oxidase activity

Cellular location - intracellular



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Ovarian reactive oxygen species (ROS) are believed to regulate ovulation in mammals, but the details of ROS production in follicles and the role of ROS in ovulation in other species remain underexplored. In Drosophila ovulation, matrix metalloproteinase 2 (MMP2) is required for follicle rupture by degradation of posterior follicle cells surrounding a mature oocyte. MMP2 activation and follicle rupture are regulated by the neuronal hormone octopamine (OA) and the octopamine receptor in mushroom body (OAMB). This study investigated the role of the superoxide-generating enzyme NADPH oxidase (NOX) in Drosophila ovulation. Nox is highly enriched in mature follicle cells, and Nox knockdown in these cells leads to a reduction in superoxide and to defective ovulation. Similar to MMP2 activation, NOX enzymatic activity is also controlled by the OA/OAMB-Ca(2+) signaling pathway. In addition, this study reports that extracellular superoxide dismutase 3 (SOD3) is required to convert superoxide to hydrogen peroxide, which acts as the key signaling molecule for follicle rupture, independent of MMP2 activation. Given that Nox homologs are expressed in mammalian follicles, the NOX-dependent hydrogen peroxide signaling pathway that is described in this study could play a conserved role in regulating ovulation in other species (Li, 2018).

Ovulation is a key step in animal reproduction and involves multiple endocrine, paracrine, and autocrine signaling molecules, such as progesterone, epidermal growth factors, and prostaglandins. These molecules ultimately activate proteinases that break down the ovarian follicle wall, releasing a fertilizable oocyte. Several lines of evidence indicate that reactive oxygen species (ROS) also play indispensable roles in mammalian ovulation. However, there is no genetic evidence to support an in vivo role of ROS in ovulation, and the enzymes responsible for ROS production during ovulation are still unknown (Li, 2018).

ROS are oxygen-derived, chemically reactive small molecules and include superoxide anion (O2*-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH*). The physiological generation of ROS can occur as a byproduct of aerobic metabolism or as the primary function of the family of NADPH oxidases (NOXs). NOX enzymes transfer an electron across the cell membrane from NADPH in the cytosol to oxygen (O2) in the luminal or extracellular space. This movement of an electron generates O2*-, which can be rapidly converted into H2O2 by superoxide dismutases (SODs) (Li, 2018).

The mammalian NOX family comprises seven members (NOX1-5 and DUOX1-2), which have marked differences in tissue distribution and play a variety of physiological roles. Members of this family are also expressed in mammalian ovaries. Nox4 and Nox5, for example, are expressed in human granulosa cells. NOX4 and its accessory proteins in human granulosa cells show age-dependent reductions in protein expression, which correlates with low fertility. Importantly, pharmacological inhibition of NOX enzymes blocks follicle-stimulating hormone-induced oocyte maturation in mouse cumulus-oocyte complex in vitro. Despite these observations, a role for NOX in mammalian ovulation has not been demonstrated (Li, 2018).

The NOX family of enzymes is evolutionarily conserved across species. The Drosophila genome contains one Nox gene encoding NOX and one Duox gene encoding DUOX. DUOX has an additional peroxidase domain and has been well studied in gut-microbe interaction, wing formation, and wound healing. Much less is known about Nox. Earlier work reported that Nox regulates ovarian muscle contraction, which somehow influences ovulation. However, the mechanism of NOX regulation of ovulation and the cellular localization of NOX in Drosophila remain unclear (Li, 2018).

Recent work challenges the concept that ovulation is controlled by ovarian muscle contraction in Drosophila. Instead, Drosophila ovulation involves active proteolytic degradation of the follicle wall and follicle rupture and shares much in common with mammalian ovulation. Like in mammals, each oocyte in Drosophila is encapsulated in a layer of somatic follicle cells to form an egg chamber, which develops through 14 distinct stages to become a mature follicle (stage-14 egg chamber) in ovarioles. In mature follicles, the zinc finger transcription factor Hindsight (HNT) induces the expression of matrix metalloproteinase 2 (MMP2) in posterior follicle cells and octopamine receptor in mushroom body (OAMB) in all follicle cells. During ovulation, octopamine (OA) is released from neuron terminals in the ovary and binds to its receptor OAMB in stage-14 follicle cells. OAMB receptor activation causes an increase in intracellular calcium that activates MMP2 enzymatic activity, which breaks down posterior follicle cells and induces follicle rupture. Strikingly, the entire process of follicle rupture can be recapitulated ex vivo by culturing isolated mature follicles with OA in the absence of ovarian muscles and oviducts. This work casts doubt on the proposed involvement of ovarian muscles in follicle rupture/ovulation (Li, 2018).

This study investigated the role of Nox in Drosophila ovulation. Surprisingly, it was found that ovarian muscle Nox does not play a major role in ovulation but rather that Nox is enriched in mature follicle cells and is essential for follicle rupture/ovulation. OA/OAMB-Ca2+ signaling activates NOX enzymatic activity to produce extracellular O2*-, which is converted into H2O2 by an extracellular SOD3. These results suggest that NOX-produced ROS in mature follicles play a conserved role in regulating follicle rupture/ovulation across species (Li, 2018).

Ovarian ROS are indispensable for ovulation in mice. However, the site of production of ROS is unknown and it is unclear whether ROS play a conserved role in ovulation across species. This study provides genetic evidence that follicular ROS are required for ovulation in Drosophila. NOX, whose activity is regulated by follicular adrenergic signaling, regulates follicle rupture and ovulation by producing O2*- in the extracellular space of mature follicle cells. In addition, the data suggest that an extracellular SOD3 converts this O2*- into H2O2, which is the key signaling molecule responsible for regulating follicle rupture. H2O2 can partially mimic LH in regulating cumulus expansion and gene expression in mammalian follicles. It is thus plausible that H2O2 plays a conserved role in regulating follicle rupture/ovulation from insects to mammals (Li, 2018).

Members of the NOX family are also expressed in mouse and human granulosa cells and are functional in producing ROS. Norepinephrine, the mammalian counterpart of OA, is highly enriched in human follicular fluid and causes ROS generation in human granulosa cells. It will be interesting to determine whether norepinephrine plays a similar role as OA in generating ROS through regulating NOX activity during follicle rupture/ovulation in mammals (Li, 2018).

Why would Drosophila mature follicles use NOX to generate ROS during follicle rupture? ROS can be generated through the mitochondrial respiratory chain and membrane-bound NOX family enzymes, as well as by a host of intracellular enzymes, such as xanthine oxidase, cyclooxygenases, cytochrome p450 enzymes, and lipoxygenases that produce ROS as part of their normal enzymatic function. As high-level cytoplasmic ROS are detrimental to cell function and viability, limiting O2*-/H2O2 production in the extracellular environment may be essential for cell viability and function. This is consistent with the finding that overexpression of Sod1, which presumably produces extra-cytoplasmic H2O2, led to a disruption in follicle rupture and egg laying. Interestingly, Nox-knockdown follicles overexpressing Sod1 had normal follicle rupture, likely due to compensation of NOX-generated H2O2 by intracellularly produced H2O2, whereas bathing Nox-knockdown follicles in H2O2 did not rescue the defect in OA-induced follicle rupture. These findings suggest that local ROS production is essential for cellular physiology, while global ROS may be detrimental (Li, 2018).

Interestingly, Sod3 knockdown alone was sufficient to cause follicle rupture defects in Drosophila, yet mice lacking SOD3 are healthy and fertile. It is possible that SOD1 can compensate for the loss of SOD3 in mouse follicles, as mice lacking SOD1 or both SOD1 and SOD3 are subfertile or infertile, respectively (Li, 2018).

This study solved a conundrum in Drosophila ovulation. Previous work demonstrated that follicle rupture requires OA/OAMB induction of MMP2 activity in posterior follicle cells. However, OA/OAMB induces a rise in intracellular Ca2+ in all mature follicle cells. What is the role of OA/OAMB-Ca2+ in nonposterior follicle cells? This work demonstrated that OA/OAMB-Ca2+ signaling activates NOX in all follicle cells to produce O2.*- and H2O2, which are important for follicle rupture. NOX-generated ROS had a minimal effect on MMP2 activity, implying that these ROS regulate an independent pathway that is required for follicle rupture. Further studies should test whether region-specific Nox knockdown, such as only in nonposterior follicle cells, causes a follicle rupture defect (Li, 2018).

The targets of H2O2 in regulating follicle rupture are still unknown. Biological redox reactions catalyzed by H2O2 typically affect protein function by promoting the oxidation of cysteine residues. The best-characterized examples of H2O2-mediated signal transduction include several protein tyrosine phosphatases in growth factor signaling pathways, such as platelet-derived growth factor, epidermal growth factor (EGF), insulin, and B cell receptor signaling. Oxidation of the cysteine residue in the active-site motif of these phosphatases reversibly inactivates phosphatase activity and promotes growth factor signaling. The timing of H2O2 production and follicle rupture makes it unlikely that H2O2 promotes follicle rupture in Drosophila follicle cells by regulating growth factor signaling. The peak production of O2*- (and presumably of H2O2) is ~30-40 min after OA stimulation, which coincides with the beginning of follicle rupture. There is not enough time to allow growth factor signaling-mediated transcription and translation to occur before rupture happens. Alternatively, H2O2 is also involved in the activation of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, possibly through direct oxidation of a cysteine residue that prevents the inhibition of catalytic domain by the prodomain of the enzyme. The idea is favored that NOX-generated H2O2 activates ADAM or other proteinases to regulate follicle rupture in addition to MMP2 activation. Microarray and RNA-sequencing analysis identified multiple proteinases that are up-regulated in Drosophila follicle cells during ovulation, and at least six different proteinases have been suggested to be involved in mammalian ovulation. Recent bioinformatics and large-scale proteomic analyses have predicted >500 proteins containing redox-active cysteine residues, some of which could serve as the downstream effectors of H2O2 for follicle rupture (Li, 2018).

p38b and JAK-STAT signaling protect against invertebrate iridescent virus 6 infection in Drosophila

The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. This study reports that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection (West, 2018).

This study shows that infection of Drosophila with the DNA virus IIV-6 triggers a protective p38b-dependent response. While previous work has demonstrated that Drosophila p38b is critical for survival to bacterial or fungal infections and affects the tolerance to bacterial infections, this is the first time p38b has been linked to antiviral defenses. Critical targets for p38b for the protection against IIV-6 infection are the unpaireds, a family of three IL-6-like genes clustered together on Chromosome X. The genetic data presented in this study argue that the three Unpaireds function together, in a partially redundant manner, to activate the JAK-STAT pathway following IIV-6 infection, thereby driving Tot gene expression. The JAK-STAT pathway also protects against IIV-6 infection, although the role of the Tots in antiviral defense requires more study. These results also imply that p38b is activated following IIV-6 infection. While the mechanisms leading from virus infection to p38 activation are unclear, they likely involve ROS-mediated signaling as the induction of TotA expression is potently blocked by an NADPH oxidase inhibitor and require the Nox gene. This is reminiscent of the activation of p38a by ROS generated from apoptotic cells in models of tissue regeneration (West, 2018).

Interestingly, p38b has also been shown to provide tolerance to Salmonella typhimurium infections, promoting survival of the host without reducing bacterial burden. This study suggested that p38b contributes to tolerance by enabling hemocyte enlargement, and hence, engulfment of larger quantities of bacteria. In the context of IIV-6 infection, p38b could be acting to promote engulfment of infected and damaged cells, thereby providing a repair mechanism to enable the animals to better tolerate and limit virus infection. Future studies will be necessary to probe all the roles of p38b in antiviral defense (West, 2018).

Although the data presented in this study demonstrate that the JAK-STAT pathway is protective against IIV-6 infection, the protective mechanisms require further study. In the case of the RNA virus DCV, the JAK-STAT pathway is also protective, possibly through the induction of vir-1. However, the JAK-STAT pathway is not broadly antiviral and vir-1 was not induced by IIV-6. Curiously, a previous study examining the role of the JAK-STAT pathway during IIV-6 infection, using one particular hypomorphic allelic combination hopscotch (JAK), concluded that hopscotch (and by inference the JAK-STAT pathway) was not involved in protecting flies against IIV-6 infection. The current data, with multiple RNAi lines targeting stat92E, as well as the S2 cell based results with RNAi targeting domeless, hopscotch, and stat92E, demonstrate a consistent and reproducible role for this pathway in the response to and survival from IIV-6 infection. These contradictory outcomes may be due to differences in alleles used or dose delivered (West, 2018).

The Tots are intriguing candidates for JAK-STAT induced antivirals. They are rapidly evolving with evidence of positive selection, typical for immune effectors. However, the Tots have not yet been demonstrated to provide direct antimicrobial activity. To date, this study has been unable to demonstrate any antiviral activity for the Tots. In particular, over-expression of TotA resulted in reduced survival following IIV-6 infection and no change in viral titers, consistent with the previously reported general toxicity caused by over expression of this gene. Further studies, examining all six of the IIV-6 induced Tots, with both loss- and gain-of-function approaches, will be necessary to more fully examine this possibility (West, 2018).

The sensitivity of STAT knockdowns to IIV-6 infection argues that JAK-STAT signaling is an important antiviral target of p38b. However, other p38b targets are also possible. For example, an established target of p38b is the heat shock response. In the context of bacterial and fungal infections, p38b is known to regulate Heat shock factor (Hsf) expression and the induction of heat shock proteins (Hsps). In addition, another report has shown that Hsf protects flies against both RNA and DNA viral infections. Together, these results suggest that the antiviral effects of p38b could be mediated, at least in part, through Hsf and Hsps. Indeed, Hsf mutant flies display an increased rate of death after IIV-6 infection. It will be interesting to learn if the heat shock response is activated by p38b following IIV-6 infection, and how this response interacts with JAK-STAT dependent viral protection (West, 2018).

Successful host defenses detect multiple characteristics of an invading pathogen. For example, cellular damage is one common indicator of pathogenic infection that can be sensed by the innate immune system. In mammals, several danger-associated molecular patterns (DAMPs) have been characterized, including HMGB1, F-actin, and histones. Likewise, a recent report examining a Drosophila model of sterile injury demonstrated that extracellular actin activates JAK-STAT signaling. In this paradigm, detection of extracellular actin, via an unknown receptor, triggered Nox-dependent ROS generation, the activation of Src42A and Shark (Syk homolog), and induction of unpaireds and eventually Tots. This pathway is very similar to that reported in this study, although p38b was not examined in this actin-DAMP, and suggests that IIV-6 infection may cause cellular damage, rupture and the release of actin, which in turn triggers ROS production, unpaired expression, JAK-STAT signaling and the induction of Tots. Formally testing this model will be facilitated by the identification of an extracellular actin receptor (West, 2018).

In summary, this study has found a novel role for Drosophila p38b in protecting against DNA virus infection. Virus infection leads to p38b dependent responses, including the induction of the JAK-STAT activating cytokines, the Unpaireds, and the induction of downstream target genes such as the Tots. Based on the analysis of viral load, the p38b pathway appears to function primarily by increasing tolerance to IIV-6, as viral loads were not altered in the p38b strain. Whether the Tots contribute to this tolerance and, more generally, whether p38b induces a directly antiviral response, or relies entirely on the Unpaired and JAK-STAT signaling for its ability to tolerize against this viral infection will be probed in future studies (West, 2018).

Microbiota-derived lactate activates production of reactive oxygen species by the intestinal NADPH oxidase Nox and shortens Drosophila lifespan

Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. This study found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. These findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies (Iatsenko, 2018).

The epithelial surfaces of most metazoan organisms are inhabited by complex microbial communities. The composition of these microbial communities is determined by an intricate interplay of genetic and environmental factors. Changes in healthy microbiota composition, referred to as commensal dysbiosis, have been associated with pathologies like inflammatory bowel disease, obesity, diabetes, neurological disorders, chronic inflammation, and cancer. However, the vast diversity of mammalian microbiota and genetic complexity of the immune system are major obstacles to clearly establishing mechanistic links between host immune genotype, microbiota structure, and disease phenotype (Iatsenko, 2018).

Because of the simplicity of its microbiota and physiological similarity with the mammalian intestine, the Drosophila gut is a model of choice to study human intestinal pathophysiology. Studies using this model have provided insights into innate immunity signaling, host-commensal interactions, and epithelial homeostasisduring aging. Drosophila harbors a microbiota composed of 5 to 30 bacterial species, dominated by the genera Acetobacter and Lactobacillus. Although many Drosophila commensals inhabit the gut transiently and are constantly replenished from food, they affect various aspects of host physiology ranging from the promotion of larval growth to the defense against pathogens. As in humans, dysbiosis in flies is associated with disruption of gut homeostasis, inflammation, and reduced lifespan, highlighting the importance of maintaining healthy microbiota composition and abundance (Iatsenko, 2018).

Several host mechanisms restrict growth of both symbiotic and pathogenic bacteria in the Drosophila gut. Acid secretion by V-ATPases of the copper cell region in the middle midgut has been shown to eliminate most intestinal bacteria, while a chitinous barrier, the peritrophic matrix, shields epithelial cells from invading bacteria. Moreover, two inducible host defense mechanisms control both pathogens and microbiota in the gut: antimicrobial peptides (AMPs) and reactive oxygen species (ROS). Two ROS-producing enzymes, the NADPH oxidases Duox and Nox, have been implicated in the control of intestinal microbes in Drosophila. The dual oxidase Duox produces microbicidal ROS in response to uracil released by pathogenic bacteria. Nox produces ROS in response to commensal bacteria, such as L. plantarum, but how Nox is activated is not yet known. ROS not only eliminate ingested pathogens but also damage enterocytes, thereby promoting the compensatory proliferation of intestinal stem cells (Iatsenko, 2018).

In addition to triggering ROS, ingested bacteria activate the expression of several antimicrobial peptide genes in specific domains along the digestive tract. This response is initiated when DAP-type peptidoglycan from Gram-negative bacteria is sensed by the transmembrane recognition receptor PGRP-LC in the ectodermal parts of the gut or by the intracellular receptor PGRP-LE in the midgut. PGRP-LC and PGRP-LE then recruit the adaptor IMD to finally activate the NF-κB-like transcription factor Relish. The gut antibacterial response is kept in check by several negative regulators of the IMD pathway, notably by enzymatic PGRPs such as PGRP-LB and PGRP-SC that scavenge peptidoglycan. Flies lacking these negative regulators show excessive, deleterious local and systemic immune activation (Iatsenko, 2018).

The IMD pathway also shapes the commensal community structure in the intestine. For example, PGRP-LC and relish flies with defects in the IMD pathway are short lived and exhibit increased bacterial loads in their guts upon aging. Chronic over-activation of the IMD pathway is also associated with microbiota dysbiosis, characterized by the expansion of antimicrobial peptide-resistant pathobionts. However, the mechanism whereby immune dysfunction causes commensal dysbiosis and leads to age-related pathologies and lifespan reduction is not fully understood (Iatsenko, 2018).

Previously identified Drosophila PGRP-SD as a secreted pattern recognition receptor that functions upstream of PGRP-LC to enhance IMD pathway activation during systemic infection (Iatsenko, 2016). In contrast to canonical mutants of the IMD pathway, a null mutation in PGRP-SD reduces but does not abolish the immune response, providing a sensitive tool to study the IMD pathway. This study used the sensitized PGRP-SD background to investigate the role of the IMD pathway in the control of intestinal homeostasis during infection and aging. Specifically, it was asked whether PGRP-SD was required to maintain a stable commensal composition and whether loss of PGRP-SD would lead to intestinal dysbiosis and dysplasia (Iatsenko, 2018).

The mucosal immune system uses multiple complex mechanisms to maintain a balance between preserving a beneficial microbiota and eliminating pathogens. This dynamic between immune system and microbiota undergoes age-related changes in animals, including humans. In Drosophila, aging is associated with a series of hallmarks: a higher microbiota load, an increased immune response, elevated ROS, dysplasia, loss of compartmentalization, and rupture of barrier permeability. Interestingly, precocious intestinal senescence equally affects mutants with immune over-activation (e.g., Caudal, PGRP-LB) and immune deficiency (Relish, Foxo). However, causal relationships between immune dysfunction, dysbiosis, and dysplasia are not clearly established. This study used PGRP-SDsk1 mutant flies with reduced immune reactivity as a tool to characterize a pathway linking immune deficiency to precocious intestinal aging. Immune-deficient flies were found to carry increased loads of the dominant microbiota member, Lp, which led to a higher release of lactate/lactic acid. This bacterial metabolite not only acidified the intestine, but also stimulated ROS production by NOX, which triggered precocious aging. This model is appealing for two reasons. First, it does not involve a change in the microbiota composition per se, but rather an overgrowth of a common, preexisting microbiota member. Second, it shows how a bacterial metabolite, lactate, when processed by the host epithelia, drives ROS production and intestinal senescence. This model is likely to apply to other contexts, including mammalian gut microbiota (Iatsenko, 2018).

Previously, PGRP-SD was identified as a major immune sensor implicated in systemic immunity. The present study uncovered the central function PGRP-SD plays in intestinal immunity, notably by inducing an efficient immune response to pathogens while promoting tolerance to microbiota. This differential response is due to its ability to control the expression of negative regulators that confer immune tolerance. By controlling the local expression of negative regulators of IMD pathway activity, PGRP-SD might prevent the systemic spread of immune activation, a function similar to that of PGRP-LE (Iatsenko, 2018).

Numerous studies have reported an increase in total microbiota load in the gut upon aging and have speculated that this change contributes to host mortality, as elimination of microbiota often prolongs lifespan. It remained unclear whether increased microbiota loads caused accelerated ageing directly or rather indirectly, by inducing chronic immune activation in the gut. This study answers this question by proving that the PGRP-SDsk1 mutant, which has increased microbiota loads but is incapable of chronic immune activation, still displays hallmarks of accelerated aging. The findings unravel a direct causal mechanism linking increases in bacterial loads to gut senescence. Specifically, this study showed that aging PGRP-SDsk1 mutants lose control over their microbial communities, resulting in increased microbiota loads dominated by Lactobacillus. The Lp-derived metabolite lactic acid/lactate stimulated ROS production by the NADPH oxidase Nox. This model is supported by the observation that a lactic acid-deficient Lp strain did not cause any precocious aging in PGRP-SDsk1 mutants. Moreover, feeding flies with lactic acid was sufficient to recapitulate all the aging hallmarks caused by Lp. Importantly, lactate produced by the symbionts needed to enter and be processed in host cells to activate NOX. This was supported by the observation that inactivating the host's lactate dehydrogenase or the lactate transporters in enterocytes suppressed Lp- or lactic acid-mediated dysplasia and lifespan shortening (Iatsenko, 2018).

In other contexts, dysbiosis with a change in microbiota composition has been recognized as a contributing factor to epithelial dysplasia, immune senescence, and age-related mortality. For example, flies with reduced Caudalexpression and high IMD pathway activity favor the growth of the pathobiont Gluconobacter EW707, which drives host mortality. In contrast, no changes were found in microbiota composition between PGRP-SDsk1 mutant and wild-type flies, excluding the possibility that immune defects in PGRP-SDsk1 mutants favor the selection of pathobionts. Thus, excessively increased loads of an otherwise beneficial Lp symbiont in PGRP-SDsk1 flies seem to be solely responsible for the lifespan shortening (Iatsenko, 2018).

Of note, mammalian PGRPs have also been implicated in the regulation of microbiota composition. Mice deficient for any of the four PGRPs (Pglyrp1-4) are more sensitive to colitis than wild-type mice due to a more inflammatory microbiota. This points to a conserved role of PGRPs as modulators of host-microbe interactions in the gut (Iatsenko, 2018).

Moreover, the finding that a commensal bacterium becomes detrimental in the immunocompromised background holds true for mammals as well. For instance, Lactobacilli that are beneficial members of human gut microbiota were associated with diseases like D-lactic acidosis, bacteremia, endocarditis, and localized infections (Iatsenko, 2018).

Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster

Damage associated molecular patterns (DAMPs) are released by dead cells and can trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. This study reports that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes evolution of adaptive immunity (Srinivasan, 2016).

Trauma, burns, ischemia, strenuous exercise, all induce a sterile inflammatory response. It is likely that this response evolved to clear cell debris, promote tissue repair and maintain tissue sterility but, if uncontrolled, it can lead to (aseptic) shock and, in some cases, death. The prevailing notion is that sterile inflammation is initiated by pro-inflammatory signals that are released by damaged cells. These include intracellular components that are exposed when cells lose their membrane integrity, such as ATP, uric acid, RNA and DNA, collectively known as damage-associated molecular patterns (DAMPs). The universe of DAMPs and their receptors, as well as the mechanisms regulating DAMP responses, remains underexplored. This is partly because early research in this area was tainted by issues of microbial contamination and because immunologists have often focussed on sterile inflammation from the narrow perspective of adaptive immunity. However, it is probable that responses to DAMPs, like responses to microbes, pre-date the vertebrate evolution of T and B cells and have an early metazoan origin, much like the clearance of dead cells. Therefore, the study of invertebrate responses to DAMPs could offer a different perspective into the induction of sterile inflammation, akin to how research into insect immunity to infection led to the identification of Toll signalling and paved the way to the discovery of an analogous pathway in vertebrates (Srinivasan, 2016).

The immune system of Drosophila melanogaster has been widely studied in the context of infection. It consists of a cellular and a humoural arm, in addition to cell-intrinsic antiviral RNAi responses. The cellular arm is made up of three macrophage-like types of cells, collectively termed haemocytes. The humoural immune response relies on antimicrobial peptides (AMPs) that are synthesised in the fat body (the fly equivalent of the liver) and then secreted into the haemolymph to provide systemic protection from bacteria and fungi. The production of AMPs is regulated by two different pathways. The Toll pathway is activated by peptidoglycan fragments of Gram-positive bacteria, fungal β-glucans, and pathogen-derived protease activity in the haemolymph. The Imd pathway is activated by peptidoglycan fragments from Gram-negative bacteria. Activation of either pathway results in the translocation of distinct NF-κB family transcription factors into the nucleus and the subsequent synthesis of AMPs best suited to neutralise the type of microorganism detected. A third pathway contributing to Drosophila humoural immunity involves Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling. In contrast to the Toll and Imd pathway, the JAK/STAT pathway has not yet been shown to be directly induced by sensors of invading microorganisms. However, it has been implicated in resistance to as well as tolerance to viral infections. Notably, the JAK/STAT pathway is activated by different types of stresses (e.g. heat, mechanical pressure, oxidative stress or UV irradiation). All of these insults likely result in cell death suggesting the possibility that JAK/STAT pathway activation might be triggered by DAMPs rather than microbes (Srinivasan, 2016).

The JAK/STAT pathway is elicited by cytokines of the Unpaired (Upd) family -- Upd1, Upd2 and Upd3 -- all of which serve as ligands for the only JAK/STAT-coupled receptor in Drosophila, Domeless (dome). The binding of Upds induces Domeless dimerization and activation of a single JAK (termed Hopscotch). Activated Hopscotch proteins phosphorylate one another allowing for recruitment of the single Drosophila STAT family transcription factor, STAT92E. The latter is then phosphorylated by Hopscotch, resulting in dimerisation and translocation into the nucleus. STAT92E dimers bind to the promoters of their target genes including, amongst others, ones encoding proteins involved in viral resistance, as well as proteins of the Turandot family such as Turandot M (TotM). The exact function of Turandot family proteins is not known but they have been controversially argued to be linked to stress resistance. Besides a role in host defence, the JAK/STAT pathway has also been linked to energy metabolism and regenerative processes, for example in the gut. The involvement of JAK/STAT signalling in regeneration is particularly interesting given the role of DAMPs in contributing to tissue repair (Srinivasan, 2016).

Previously work has identified DNGR-1 (also known as CLEC9A) as a vertebrate-restricted innate immune receptor dedicated to DAMP recognition (Sancho, 2009). DNGR-1 is phosphorylated by Src family kinases and then signals via Syk although it does not induce inflammation. Rather, DNGR-1 is expressed by dendritic cells (DCs) and signals to favour cross-presentation of antigens from dead cells, contributing to CD8+ T cell responses to cytopathic infections and, possibly, tumours. The DAMP recognised by DNGR-1 is F-actin, the polymer of G-actin that provides higher eukaryotic cells with structural integrity (Ahrens, 2012; Zhang, 2012). Actin is an ideal DAMP given that it is extremely conserved (90% identity between yeast and humans) and highly abundant and ubiquitous within all eukaryotic cells but absent from extracellular fluids. It was therefore hypothesised that released actin constitutes an evolutionarily-conserved DAMP whose detection might involve a signalling pathway conserved from flies to mammals. This would be analogous to the conservation of the Toll signalling pathway (albeit not the upstream receptors) in the Drosophila and vertebrate response to fungi and bacteria. This study shows systemic administration of actin to Drosophila selectively triggers a JAK/STAT response and that this requires the fly homologues of Src and Syk. The data therefore reveal an evolutionarily-conserved tyrosine kinase-based pathway for recognising damage through sensing of released or exposed actin (Srinivasan, 2016).

Dysregulated and/or chronic inflammation, often of sterile origin, is increasingly recognised as a contributing factor to a vast range of human diseases, from cancer to neurodegeneration. Furthermore, because injury and infection often overlap, understanding of immunity necessitates a consideration of the interplay between the processes that detect pathogen invasion and those that sense tissue damage. The study of invertebrate responses to DAMPs might therefore lead to a new understanding of sterile inflammation and the identification of conserved elicitors, detectors and signaling pathways that are utilised across evolution to detect loss of cell integrity (Srinivasan, 2016).

Previous work has shown that actin, one of the most abundant and conserved proteins in eukaryotic cells, acts as a DAMP in mouse and humans, binding to DNGR-1, a Src and Syk-coupled dead cell receptor expressed on DCs. This study provides evidence that actin is also a DAMP in Drosophila melanogaster, triggering a response that, like in vertebrates, requires Syk and Src family kinases. The presence of extracellular actin in the haemolymph of Drosophila elicits a reaction in the fat body via Shark and Src42A, whose activation depends on reactive oxygen species (ROS) generated by the NADPH oxidase Nox. Consistent with these data, ROS generation by NADPH oxidases is a highly conserved response to wounding and has been shown to directly activate Lyn/Src42A in zebrafish and Drosophila through oxidation of a single redox-sensitive cysteine residue (Srinivasan, 2016).

In contrast to DNGR-1 dependent recognition, the fly response to extracellular actin is elicited equally by G- and F-actin, does not require phagocytes but the fat body and its function is not to prime adaptive immunity, which is absent in invertebrates. Rather, it is coupled to production of Upd3 cytokine, which acts in an autocrine and paracrine manner to induce Domeless signalling via STAT and to cause the induction of STAT-responsive genes, the products of which are released into the haemolymph. This systemic inflammatory-like response involving cytokine amplification and the fat body is reminiscent of the acute phase response in mammals, which can be triggered by infection or trauma and leads to the production of cytokines such as IL-6 that act on the liver (mammalian equivalent of the fat body) to cause production of acute phase proteins. These are secreted into the plasma to regulate multiple processes such as host defence, coagulation, vascular permeability and metabolism (Medzhitov, 2010). Similarly, the Drosophila fat body response to actin results in secretion into the haemolymph of proteins that may regulate multiple aspects of fly physiology that coordinately impact resistance or tolerance to insult. However, it is important to note that while some components of the extracellular actin-sensing circuitry are conserved between flies and mammals (Shark, Src42A and ROS), others are not (DNGR1, cross-presentation, dendritic cells). These differences suggest that DAMPs can be more conserved than their receptors or the responses they evoke. This is akin to pathogen-associated molecular patterns (PAMPs) such as, for example, lipopolysaccharide (LPS), a hallmark of Gram-negative bacteria. The sensing of LPS is conserved in plants, protists and animals, but the relevant receptors and subsequent responses diverge depending on the host. Similarly, peptidoglycans and β-glucans are used in both flies and mammals to signify bacterial or fungal presence, yet are detected by different receptors that, nevertheless, can couple to conserved signalling pathways (Srinivasan, 2016).

The JAK/STAT pathway in Drosophila can be induced by mechanical pressure, heat shock, dehydration, cytopathic infection, septic wounds and other traumas. How such seemingly disparate stimuli trigger a single pathway is puzzling. However, a common denominator in all these settings is cell death and it has been speculated that STAT activation might therefore occur in response to DAMP release. The current data support that notion and suggest that actin is a potent DAMP for triggering the JAK/STAT pathway. Notably, pathogen infection in Anopheles gambiae and Drosophila melanogaster has been shown to lead to the release of actin into the haemolymph, where it can act as an antibacterial or antiparasitic agent. Therefore, actin release may serve as a two-pronged defense mechanism, both directly as an antimicrobial and indirectly by activating a systemic JAK/STAT response (Srinivasan, 2016).

The role of the systemic JAK/STAT response is unclear at present. Despite being commonly used as a marker of STAT activation, the function of Tot and Tep proteins in Drosophila is unknown. Nevertheless, genetic loss-of-function studies have implicated JAK/STAT signaling in resistance and/or tolerance to viral, bacterial and parasitoid infections. Furthermore, the JAK/STAT pathway has a well-established role in maintenance of fly intestinal homeostasis, both at steady state and following infection or injury. Given these precedents, attempts were made to investigate the role of the inducible actin-triggered JAK/STAT circuit by injecting actin into flies prior to challenge with viruses (Flock house virus, Drosophila C virus, Sindbis virus and Cricket paralysis virus) or bacteria (Erwinia carotovora, Escherichia coli, Micrococcus luteus and Listeria monocytogenes) but failed to find an effect on either resistance or tolerance to infection. Similarly, in models of stress or injury (starvation, heat shock, irradiation, paraquat feeding and a recently-described model of concussion), no evidence was found of protection or susceptibility afforded by actin pre-injection. Finally, no effect was found of actin injection on fat body metabolism. The failure to find a system in which prior upregulation of STAT target genes by exogenous actin leads to a difference in outcome is a current experimental limitation. However, it might reflect the fact that STAT activation is already induced to sufficient levels in those models in response to actin released from dying cells. Consistent with this notion, septic injury was observed to lead to a rapid increase in actin levels within the haemolymph. In such a situation, additional induction of the STAT pathway by actin pre-injection may not confer additional protection or tolerance. Reinforcing this notion is a recent study showing that loss of basal Diedel levels leads to reduced tolerance to Sindbis virus, yet the upregulation of Diedel levels that takes place during infection is itself dispensable. Unfortunately, loss-of-function experiments to assess the effect of released actin on different challenges are not feasible because actin is essential for viability. Surrogate loss-of-function experiments, such as examining the role of Nox and Src42A or Shark in the fat body in the context of infection or injury, have not been reported and their interpretation is complicated by the pleiotropic effects of those proteins. Nevertheless, the finding that actin is released into the haemolymph upon septic injury and that this induces JAK/STAT activation dependent on fat body expression of Src42A and Nox may suggest that previous reports of septic injury-induced STAT activation can be partially ascribed to extracellular actin (Srinivasan, 2016).

The identity of the putative receptor that recognises extracellular actin in Drosophila remains unknown. The requirement for Upd3 rules out the possibility that actin serves as a direct ligand for Domeless, a conclusion further supported by the fact that actin does not induce TotM upregulation in various Drosophila cell lines that respond to Upd cytokines in vitro. Therefore, the simplest interpretation of the data is that Upd3 is synthesised by fat body cells that detect extracellular actin via a sensor(s) that couple(s) to a Nox-Src42A-Shark cascade. By analogy with other receptors that engage a Syk-dependent pathway, that sensor might be an ITAM- or hemITAM-bearing receptor or one that associates in trans with an ITAM-containing signalling chain. Interestingly, in Drosophila responses to wounding and in the clearance of axonal debris and neuronal cell corpses, one such receptor is Draper, a member of the Nimrod family and orthologue of C. elegans Ced1. Draper contains an ITAM that is phosphorylated by Src42A. However, Draper was found to be dispensable for TotM induction in response to actin injection. Similarly, no role was found for Nimrod C1, C4 and the scavenger receptor CD36. Whether these data indicate the activity of an unknown receptor, multiple redundant receptors or an indirect sensing mechanism, akin to the activation of the vertebrate NLRP3 receptor, will need to be investigated (Srinivasan, 2016).

In sum, these data suggest that extracellular actin released by dead cells induces a response in Drosophila that requires signalling in the fat body via the non-receptor tyrosine kinase, Shark, and the Src family kinase, Src42A. This pathway leads to production of Upd cytokines that act in an autocrine and paracrine manner to induce Domeless signalling via STAT and cause induction of STAT-responsive genes. Thus, the presence of actin in the extracellular space triggers a response previously associated with wounding and dead cell clearance, indicating that actin exposure acts as an ancient sign of tissue damage and that actin constitutes an evolutionarily-conserved DAMP. The notion that actin exposure can act as a universal sign of cell damage might apply more generally to other cytoskeletal proteins (Srinivasan, 2016).

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. This study reports that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. These data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential (Jones, 2013).


Functions of Nox orthologs in other species

An interaction between Scribble and the NADPH oxidase complex controls M1 macrophage polarization and function

The polarity protein Scribble (SCRIB) regulates apical-basal polarity, directional migration and tumour suppression in Drosophila and mammals. This study reports that SCRIB is an important regulator of myeloid cell functions including bacterial infection and inflammation. SCRIB interacts directly with the NADPH oxidase (NOX) complex in a PSD95/Dlg/ZO-1 (PDZ)-domain-dependent manner and is required for NOX-induced reactive oxygen species (ROS) generation in culture and in vivo. On bacterial infection, SCRIB localized to phagosomes in a leucine-rich repeat-dependent manner and promoted ROS production within phagosomes to kill bacteria. Unexpectedly, SCRIB loss promoted M1 macrophage polarization and inflammation. Thus, SCRIB uncouples ROS-dependent bacterial killing activity from M1 polarization and inflammatory functions of macrophages. Modulating the SCRIB-NOX pathway can therefore identify ways to manage infection and inflammation with implications for chronic inflammatory diseases, sepsis and cancer (Zheng, 2016).

Downregulation of SCRIB expression in a macrophage cell line, RAW 264.7, significantly impaired phorbol 12-myristate 13-acetate (PMA)-induced generation of ROS. In addition, primary macrophages were used from an inducible SCRIB knockdown mouse model where SCRIB short hairpin RNA (shRNA) expression was controlled by a tetracycline-response element (TRE) at the COLA1 locus (SCRIB ishRNA). Reverse tetracycline transactivator (rtTA) expression from a ROSA26 promoter facilitated doxycycline (Dox)-inducible expression of SCRIB ishRNA and GFP in all cells, including isolated bone-marrow-derived macrophages (BMMs). Inducible loss of SCRIB did not affect BMM differentiation as monitored by expression of CD11b/F4/80 and cell morphology. Both PMA and lipopolysaccharide (LPS) induced ROS in wild-type cells and in SCRIB ishRNA cells in the absence of Dox stimulation. Dox-induced knockdown of SCRIB significantly impaired PMA- or LPS-induced ROS. In addition, PMA-induced ROS was significantly low in primary neutrophils from Dox-treated mice, demonstrating a role for SCRIB during ROS generation in primary myeloid cells (Zheng, 2016).

Apart from myeloid cells, in mouse embryo fibroblasts (MEFs) derived from SCRIB ishRNA mice platelet-derived growth factor (PDGF)-induced generation of ROS and tyrosine phosphorylation of the PDGF receptor were impaired, demonstrating a role for SCRIB in regulating ROS generation in multiple contexts. The decrease in PDGFR tyrosine phosphorylation is consistent with the well-established role for ROS in inactivating tyrosine phosphatase activity and promoting tyrosine phosphorylation (Zheng, 2016).

To determine the in vivo significance, SCRIB ishRNA mice were injected with LPS intraperitoneally and ROS was monitored in real time using L-012, a luminol-based chemiluminescence probe used for detecting NADPH oxidase-derived superoxide. LPS challenge induced a dramatic and sustained increase in ROS levels in -Dox, but not in +Dox mice in vivo. The lack of LPS-induced ROS in vivo was not due to defective immune biology because cells from the thymus, spleen and bone marrow of wild-type (WT) (+Dox and -Dox) and SCRIB ishRNA (+Dox and -Dox) mice showed comparable expression of differentiation markers on dendritic, neutrophil, monocyte or B and T cells (Zheng, 2016).

Macrophages generate ROS (superoxide (O2-)) using the NOX complex. LPS, PMA and PDGF induce ROS by activating the NOX protein complex (Park, 2004; Segal, 1993). NOX-mediated generation of ROS requires activation of Rac GTPases facilitated by the GTP exchange factor β-PIX13. Since β-PIX binds directly with SCRIB, LPS-induced Rac activation was analysed in SCRIB shRNA cells. Interestingly, LPS-induced Rac.GTP levels were twofold lower in SCRIB shRNA cells compared with Luc shRNA cells (Zheng, 2016).

Tests were performed to see whether SCRIB functions as a scaffold to recruit β-PIX to the NOX complex. β-PIX co-immunoprecipitated both p22phox (a component of the NOX complex) and SCRIB in Flag-taggedβ-PIX-expressing control cells, but not in RAW 264.7 SCRIB shRNA cells. The interaction was observed both before and after PMA stimulation, with a modest increase in association post PMA stimulation (Zheng, 2016).

SCRIB is a PSD95/Dlg/ZO-1 (PDZ)-domain-containing protein and PDZ domains frequently use carboxy-terminal residues to interact with its partners. Analysis of the carboxy-terminal 10 amino acids of all members of the NOX complex showed that only the C-terminal sequence of p22phox (192-DEVV-195) conforms to a class III PDZ-binding motif (X(unspecified)-D/E-X-Ψ(hydrophobic)). Amino-terminal Flag-tagged WT p22phox, but not the p22phox lacking the C-terminal 10 amino acids (p22Δ), interacted with T7 epitope-tagged SCRIB. p22phox is a common subunit present in all of the NOX complexes (NOX1-4); accordingly, SCRIB co-immunoprecipitated with NOXO1 (a component of NOX1 and 3) and Tks5 (a component of NOX4), identifying SCRIB as a member of all NOX complexes. Both the NOX1 and NOX3 complexes require Rac.GTP for their activity, supporting a role for SCRIB in these complexes (Zheng, 2016).

Endogenous p22phox from both RAW 264.7 and primary BMMs was able to co-immunoprecipitate SCRIB. Conversely, endogenous SCRIB co-immunoprecipitated p22phox. In contrast to SCRIB-p22phox association, interaction with other components of the NOX complex, gp91phox, p67phox and p40phox, was induced on LPS stimulation in a SCRIB-dependent manner. This is consistent with previous observations that LPS stimulation induces assembly of an active NOX complex (Zheng, 2016).

To determine whether SCRIB and p22phox interact directly with each other, bacterially produced 6xHis-tag fusions were used of each of the four PDZ domains (SCRIB-PDZ1, SCRIB-PDZ2, SCRIB-PDZ3 and SCRIB-PDZ4) and GST-tag-fused p22phox C-terminal tail (186-NPIPVTDEVV-195) (p22tail). Purified SCRIB-PDZ4, but not the other PDZ domains, bound directly to purified p22tail. Next, a nuclear magnetic resonance (NMR) spectroscopy experiment was performed on 15N-labelled SCRIB-PDZ4 with unlabelled p22tail peptide (residues 186-195) to identify the binding interface. By increasing the concentration of the peptide, nine peaks were observed displaying marked peak shifts (>0.2 normalized chemical shift change) in the 1H-15N heteronuclear single-quantum coherence spectra. These perturbed residues (Leu1111, Gly1112, Ile1113, Ser1114, Ile1115, Ala1144, Ala1145, Arg1178 and Val1180) were mapped onto the previously reported three-dimensional structure of unligated SCRIB-PDZ4 (PDB: 1UJU). In contrast, a p22phox peptide lacking the carboxy tail (residues 131-185) displayed no change in the NMR spectra of SCRIB-PDZ4. A typical PDZ domain consists of six β-strands (βA-βF) and two α-helices (αA and αB). The most affected residues Leu1111 and Gly1112 are located in the well-known target-carboxylate binding loop (1110-RLGI-1113), while other perturbed residues, Ser1114 and Ile1115, reside at the βB strand and Arg1178 is located within the αB helix. The identified p22tail binding site on SCRIB-PDZ4 coincides well with the canonical target-binding pocket found in many PDZ proteins. Accordingly a p22tail-docking model structure of SCRIB-PDZ4 was generated using the structure of the Erbin PDZ domain-ErbB2 peptide complex (PDB 1MFG18) as a template. Additional tests confirmed that the PDZ4 domain of SCRIB is essential for interaction with the NOX complex and for the SCRIB-dependent production of ROS (Zheng, 2016).

NOX complex-generated respiratory burst within phagosomes is required to kill invading bacteria. To investigate whether loss of SCRIB affects the ability of phagocytes to clear bacterial infection, SCRIB ishRNA mice were challenged with an intraperitoneal injection of Staphylococcus aureus. SCRIB ishRNA - Dox mice were fourfold more effective in clearing S. aureus from both the peritoneal cavity and the lung compared with mice on a Dox diet. To rule out a role for differences in neutrophil migration, purified neutrophils and blood preparations from SCRIB ishRNA + Dox or -Dox mice were analyzed in culture; myeloid cells from +Dox mice have a fourfold decrease in S. aureus killing compared with cells from SCRIB ishRNA - Dox or WT mice, demonstrating a cell-intrinsic defect. Furthermore, both RAW 264.7 control and SCRIB shRNA cells were equally competent in internalizing pHrodo dye-conjugated S. aureus particles demonstrating that bacterial phagocytosis was unaffected by loss of SCRIB (Zheng, 2016).

Next, the relationship between SCRIB and phagosomes was investigated. Structured illumination microscopy of RAW 264.7 cells 1.0 h post S. aureus infection demonstrated co-localization of SCRIB and p22phox around S. aureus. Both the PDZ4 mutant and WT SCRIB, expressed in the background of SCRIB shRNA, were effective in localizing to the bacteria, demonstrating that SCRIB-p22phox interaction was not required for SCRIB to localize to phagosomes. In both SCRIB WT and PDZ4 rescue cell lines, SCRIB was localized at a comparable distance to bacteria (median distance between 1.5-2.0 µm), as monitored by conventional confocal image analysis. In addition to RAW 264.7 cells, SCRIB accumulated near bacteria containing phagosomes in a human macrophage cell line Thp1 expressing RFP-SCRIB and infected with FITC-S. aureus demonstrating evolutionary conservation of the mechanism (Zheng, 2016).

In both Drosophila and mammalian epithelial cells, SCRIB regulates cell polarity in a membrane localization-dependent manner. A SCRIB mutant that fails to localize to the cell membrane, Pro305 to leucine (P305L), also failed to localize near bacteria, identifying a role for membrane localization in localizing SCRIB to phagosomes. Neither PDZ4 nor SCRIBP305L expression in RAW 264.7 SCRIB shRNA cells rescued PMA-induced generation of ROS, suggesting that both interaction with p22phox and localization to the phagosome were required for ROS generation (Zheng, 2016).

Generation of ROS within the phagosome is required for the ability of macrophages to kill bacteria. RAW 264.7 Luc shRNA and SCRIB shRNA cells were infected with S. aureus and incubated with cerium chloride, which reacts with ROS in phagosomes to form an electron-dense precipitate visible by transmission electron microscopy. Electron-dense precipitates of cerium ions were found around the S. aureus-containing phagosomes in shLuc cells, but not SCRIB shRNA cells, identifying an unexpected role for SCRIB as a regulator of ROS generation within phagocytic structures (Zheng, 2016).

Whether SCRIB regulates macrophage polarization was investigated because M1 macrophages are key regulators of host defence to pathogen infection and inflammation. RAW 264.7 and primary, bone marrow-derived, cells were stimulated with a combination of interferon gamma and LPS to induce M1 or IL-4 to induce M2 macrophage polarization. Surprisingly, cells lacking SCRIB (+Dox) showed a significant increase in the tendency to polarize towards the M1 lineage, as monitored by expression of IL-1β and Il-12β messenger RNA, but a decrease in the tendency to polarize towards the M2 lineage, as monitored by Fizz-1 mRNA expression. This phenotype was rescued by re-expression of SCRIB WT, but not by expression of PDZ4 or P305L mutants, demonstrating the need for p22phox interaction and membrane localization. Thus, the impaired bacterial killing in mice lacking SCRIB is not due to a defect in M1 polarization (Zheng, 2016).

To determine whether the hyper-inflammatory response relates to enhanced cytokine production, BMMs from SCRIB ishRNA mice were cultured, stimulated with LPS for 2.0 h and assayed for mRNA expression of inflammatory cytokines using quantitative PCR arrays. Pro-inflammatory cytokines were upregulated in SCRIB ishRNA + Dox compared with SCRIB ishRNA - Dox cells (2-10-fold) on LPS challenge. Consistent with the increase in transcript levels, protein levels of the major inflammatory cytokines, IL-6 and TNF (also known as TNFα), were significantly higher in SCRIB ishRNA + Dox mice compared with -Dox conditions. Inflammatory cytokines are primarily induced by the NF-κB transcription complex. However, neither the phosphorylation of IkB-alpha nor nuclear localization of p65 was altered by SCRIB loss. ROS can oxidize Cys62 of the p50 subunit of NF-κB ROS and interfere with its DNA-binding ability. Thus, the low levels of LPS-induced ROS in SCRIB knockdown cells can paradoxically increase the DNA-binding capacity of NF-κB to DNA and promote expression of inflammatory cytokines. Consistent with this possibility, the p65 subunit of the NF-κB complex was bound at significantly higher levels on both the IL-6 and TNF promoter in cells lacking SCRIB as determined by chromatin immunoprecipitation (ChIP). However, polymerase II occupancy on these promoters was not affected by loss of SCRIB. In addition to macrophages, dendritic cells are also sensitive to differences in ROS levels. Dendritic cells with low levels of ROS show a heightened response to LPS challenge compared with those with high levels of ROS28. Bone-marrow-derived dendritic cells (BMDCs) showed an elevated expression of canonical activation markers, such as CD80, CD86 and MHCII, in the absence of SCRIB expression (+Dox) and with LPS stimulation. Thus, loss of SCRIB results in increased DC sensitivity to LPS stimulation, which potentially exacerbates the hyper-inflammatory phenotype and lethal response observed with LPS challenge of ishSCRIB + Dox mice (Zheng, 2016).

In addition to its role in the clearance of bacterial infections, NADPH oxidase is also an important regulator of inflammation. Individuals with chronic granulomatous disease, caused by mutations in members of the NOX complex, show hyper-inflammatory symptoms such as development of granuloma, Crohn's-like disease and pulmonary fibrosis. p47phox knockout mice, in addition to having a defect in clearing bacterial infection due to decreased generation of ROS, show a hyper-inflammatory response on LPS challenge. The current observations are thus consistent with the phenotypes observed in patients with a defective NOX complex and those reported for mouse models with mutations in the NOX complex, providing genetic support to the conclusion that SCRIB is a member of the NOX complex. In addition, unexpected finding is reported that SCRIB is a regulator of M1/M2 polarization. Thus, this study defines a function for SCRIB in cells of the myeloid lineage. Apart from myeloid cells, the NOX complex plays an important role in the central nervous system including microglia function, astrocyte survival, neuronal polarization, axonal growth and neurodegenerative disease. Thus, a better understanding of the SCRIB-NOX pathway can provide insights into neurological diseases, managing bacterial infection, modulating inflammation and tumour cell killing with implications for chronic inflammatory diseases, sepsis and cancer (Zheng, 2016).

Optimal ROS signaling is critical for nuclear reprogramming

Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS) signaling, this study sought to determine whether there was a role of ROS signaling in nuclear reprogramming. ROS production was examined during the reprogramming of doxycycline (dox)-inducible mouse embryonic fibroblasts (MEFs) carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) into induced pluripotent stem cells (iPSCs). ROS generation was substantially increased with the onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22(phox)-a critical subunit of the Nox (1-4) complex-decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, the data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency (Zhou, 2016).

Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, this study sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-kappaB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. This study has identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection (Olagnier, 2014).


REFERENCES

Search PubMed for articles about Drosophila Nox

Ahrens, S., Zelenay, S., Sancho, D., Hanc, P., Kjaer, S., Feest, C., Fletcher, G., Durkin, C., Postigo, A., Skehel, M., Batista, F., Thompson, B., Way, M., Reis e Sousa, C. and Schulz, O. (2012). F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells. Immunity 36(4): 635-645. PubMed ID: 22483800

Iatsenko, I., Kondo, S., Mengin-Lecreulx, D. and Lemaitre, B. (2016). PGRP-SD, an extracellular pattern-recognition receptor, enhances peptidoglycan-mediated activation of the Drosophila Imd pathway. Immunity 45(5): 1013-1023. PubMed ID: 27851910

Iatsenko, I., Boquete, J. P. and Lemaitre, B. (2018). Microbiota-derived lactate activates production of reactive oxygen species by the intestinal NADPH oxidase Nox and shortens Drosophila lifespan. Immunity 49(5): 929-942. PubMed ID: 30446385

Jones, R. M., Luo, L., Ardita, C. S., Richardson, A. N., Kwon, Y. M., Mercante, J. W., Alam, A., Gates, C. L., Wu, H., Swanson, P. A., Lambeth, J. D., Denning, P. W. and Neish, A. S. (2013). Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species. EMBO J 32(23): 3017-3028. PubMed ID: 24141879

Li, W., Young, J. F. and Sun, J. (2018). NADPH oxidase-generated reactive oxygen species in mature follicles are essential for Drosophila ovulation. Proc Natl Acad Sci U S A 115(30): 7765-7770. PubMed ID: 29987037

Medzhitov, R. (2010). Inflammation 2010: new adventures of an old flame. Cell 140(6): 771-776. PubMed ID: 20303867

Olagnier, D., Peri, S., Steel, C., van Montfoort, N., Chiang, C., Beljanski, V., Slifker, M., He, Z., Nichols, C. N., Lin, R., Balachandran, S. and Hiscott, J. (2014). Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells. PLoS Pathog 10(12): e1004566. PubMed ID: 25521078

Park, H. S., Jung, H. Y., Park, E. Y., Kim, J., Lee, W. J. and Bae, Y. S. (2004). Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B. J Immunol 173(6): 3589-3593. PubMed ID: 15356101

Sancho, D., Joffre, O. P., Keller, A. M., Rogers, N. C., Martinez, D., Hernanz-Falcon, P., Rosewell, I. and Reis e Sousa, C. (2009). Identification of a dendritic cell receptor that couples sensing of necrosis to immunity. Nature 458(7240): 899-903. PubMed ID: 19219027

Segal, A. W. and Abo, A. (1993). The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem Sci 18(2): 43-47. PubMed ID: 8488557

Srinivasan, N., Gordon, O., Ahrens, S., Franz, A., Deddouche, S., Chakravarty, P., Phillips, D., Yunus, A.A., Rosen, M.K., Valente, R.S., Teixeira, L., Thompson, B., Dionne, M.S., Wood, W., Reis, E. and Sousa, C. (2016). Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster. Elife 5. pii: e19662. PubMed ID: 27871362

West, C. and Silverman, N. (2018). p38b and JAK-STAT signaling protect against invertebrate iridescent virus 6 infection in Drosophila. PLoS Pathog 14(5): e1007020. Pubmed ID: 29746571

Zhang, J. G., Czabotar, P. E., Policheni, A. N., Caminschi, I., Wan, S. S., Kitsoulis, S., Tullett, K. M., Robin, A. Y., Brammananth, R., van Delft, M. F., Lu, J., O'Reilly, L. A., Josefsson, E. C., Kile, B. T., Chin, W. J., Mintern, J. D., Olshina, M. A., Wong, W., Baum, J., Wright, M. D., Huang, D. C., Mohandas, N., Coppel, R. L., Colman, P. M., Nicola, N. A., Shortman, K. and Lahoud, M. H. (2012). The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments. Immunity 36(4): 646-657. PubMed ID: 22483802

Zheng, W., Umitsu, M., Jagan, I., Tran, C. W., Ishiyama, N., BeGora, M., Araki, K., Ohashi, P. S., Ikura, M. and Muthuswamy, S. K. (2016). An interaction between Scribble and the NADPH oxidase complex controls M1 macrophage polarization and function. Nat Cell Biol 18(11): 1244-1252. PubMed ID: 27694890

Zhou, G., Meng, S., Li, Y., Ghebre, Y. T. and Cooke, J. P. (2016). Optimal ROS signaling is critical for nuclear reprogramming. Cell Rep 15(5): 919-925. PubMed ID: 27117405


Biological Overview

date revised: 20 July, 2019

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