Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.

Particle uptake driven phagocytosis in macrophages and neutrophils enhances bacterial clearance.

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material.

Intramacrophage ROS Primes the Innate Immune System via JAK/STAT and Toll Activation.

Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. We show a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. We find that induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system.

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila.

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival.

Gut physiology mediates a trade-off between adaptation to malnutrition and susceptibility to food-borne pathogens.

The animal gut plays a central role in tackling two common ecological challenges, nutrient shortage and food-borne parasites, the former by efficient digestion and nutrient absorption, the latter by acting as an immune organ and a barrier. It remains unknown whether these functions can be independently optimised by evolution, or whether they interfere with each other. We report that Drosophila melanogaster populations adapted during 160 generations of experimental evolution to chronic larval malnutrition became more susceptible to intestinal infection with the opportunistic bacterial pathogen Pseudomonas entomophila. However, they do not show suppressed immune response or higher bacterial loads. Rather, their increased susceptibility to P. entomophila is largely mediated by an elevated predisposition to loss of intestinal barrier integrity upon infection. These results may reflect a trade-off between the efficiency of nutrient extraction from poor food and the protective function of the gut, in particular its tolerance to pathogen-induced damage.

The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine.

The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. In this study, we have analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. We showed that p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora 15. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Duox is reduced in p38c mutant flies. Our genetic analysis shows that p38c functions in a pathway with Mekk1 and Mkk3 to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, our study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c.

Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is involved in the regulation of intestinal stem cell (ISC) activity to ensure a continuous renewal of the adult Drosophila midgut. Three ligands, Unpaired 1, Unpaired 2 and Unpaired 3 (Upd1, Upd2 and Upd3, respectively) are known to activate the JAK/STAT pathway in Drosophila. Using newly generated upd mutants and cell-specific RNAi, we showed that Upd1 is required throughout the fly life to maintain basal turnover of the midgut epithelium by controlling ISC maintenance in an autocrine manner. A role of Upd2 and Upd3 in basal conditions is discernible only in old gut, where they contribute to increased ISC abnormal division. Finally, upon an acute stress such as oral bacterial infection, we showed that Upd3 is released from enterocytes and has an additive effect with Upd2 to promote rapid epithelial regeneration. Taken together, our results show that Upd ligands are required to maintain the midgut homeostasis under both normal and pathological states.

Infection-induced host translational blockage inhibits immune responses and epithelial renewal in the Drosophila gut.

Typically, immune responses control the pathogen, while repair and stress pathways limit damage caused by pathogenesis. The relative contribution of damage to the outcome of pathogenesis and the mechanistic links between the immune and repair pathways are poorly understood. Here, we analyze how the entomopathogenic bacterium Pseudomonas entomophila induces irreversible damage to the Drosophila gut. We find that P. entomophila ingestion induces a global translational blockage that impairs both immune and repair programs in the fly gut. P. entomophila-induced translational inhibition is dependent on bacterial pore forming toxins and reactive oxygen species produced by the host in response to infection. Translational arrest is mediated through activation of the GCN2 kinase and inhibition of the TOR pathway as a consequence of host damage. Together, our study draws a model of pathogenesis in which bacterial inhibition of translation by excessive activation of stress responsive pathways inhibits both immune and regenerative epithelial responses.

Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.

Heat shock factor gene family in rice: genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses.

Binding of heat shock factors (HSFs) with heat shock element sequence is critical for the transcriptional induction of heat shock genes. Rice genome sequence shows 26 OsHsf genes out of which 25 possess various important domains noted in HSFs i.e. DNA binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES) and AHA type activation domain. OsHsf entry LOC_Os06g226100 has the oligomerization domain but lacks the above other domains. Also, there are no ESTs or full-length cDNA noted for this entry in database. Expression profiling showed that 22 OsHsf genes are induced by high temperature. Induction of 10 and 14 OsHsf genes was also noted against low temperature stress and oxidative stress, respectively. All OsHsf genes induced by oxidative stress were also induced by high temperature. On the other hand, induction of 6 and 1 OsHsf genes was noted to be exclusive to high and low temperature stresses, respectively. Seven OsHsf genes showed induced expression in response to all the three stresses examined. While in silico promoter analysis showed that OsHsf genes contain upstream regulatory elements corresponding to different abiotic stresses, there was lack of correlation noted between the in silico profiling of the elements and their corresponding transcript expression patterns. Apart from stress inducibility, EST database suggests that various OsHsf genes are developmentally regulated in diverse tissue types.