Effects of Fluticasone Propionate on Klebsiella pneumoniae and Gram-Negative Bacteria Associated with Chronic Airway Disease.

Inhaled corticosteroids (ICS) are commonly prescribed first-line treatments for asthma and chronic obstructive pulmonary disease (COPD). Recent evidence has shown that ICS use is associated with changes in the airway microbiome, which may impact clinical outcomes such as potential increased risk for pneumonia in COPD. Although the immunomodulatory effects of corticosteroids are well appreciated, whether ICS could directly influence the behavior of respiratory tract bacteria has been unknown. In this pilot study we explored the effects of fluticasone proprionate, a commonly prescribed inhaled corticosteroid, on respiratory bacteria with an expanded focus on Klebsiella pneumoniae, a species previously implicated in fluticasone-associated pneumonia in COPD. We observed significant effects of fluticasone proprionate on growth responses of K. pneumoniae, as well as other bacterial species isolated from asthmatic patients. Fluticasone-exposed K. pneumoniae displayed altered expression of several bacterial genes and reduced the metabolic activity of bronchial epithelial cells and their expression of human ß-defensin 2. Targeted assays identified a fluticasone metabolite from fluticasone-exposed K. pneumoniae cells, suggesting this species may be capable of metabolizing fluticasone proprionate. Collectively, these observations support the hypothesis that specific members of the airway microbiota possess the functional repertoire to respond to or potentially utilize corticosteroids in their microenvironment. These findings lay a foundation for novel research directions into the potential direct effects of ICS, often prescribed long term to patients, on the broader airway microbial community and on the behavior of specific microbial species implicated in asthma and COPD outcomes. IMPORTANCE Inhaled corticosteroids are widely prescribed for many respiratory diseases, including asthma and COPD. While they benefit many patients, corticosteroids can also have negative effects. Some patients do not improve with treatment and even experience adverse side effects. Recent studies have shown that inhaled corticosteroids can change the make-up of bacteria in the human respiratory tract. However, whether these medications can directly impact the behavior of such bacteria has been unknown. Here, we explored the effects of fluticasone propionate, a commonly prescribed inhaled corticosteroid, on Klebsiella pneumoniae and other airway bacteria of interest, including primary species isolated from adult asthma patients. We provide evidence of growth responses to direct fluticasone exposure in culture and further examined fluticasone's effects on K. pneumoniae, including gene expression changes and effects of fluticasone-exposed bacteria on airway cells. These findings indicate that members of the human airway bacterial community possess the functional ability to respond to corticosteroids, which may have implications for the heterogeneity of treatment response observed clinically.

Wolbachia Inter-Strain Competition and Inhibition of Expression of Cytoplasmic Incompatibility in Mosquito.

Successful field trials have been reported as part of the effort to develop the maternally transmitted endosymbiontic bacteria Wolbachia as an intervention agent for controlling mosquito vectors and their transmitted diseases. In order to further improve this novel intervention, artificially transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired cytoplasmic incompatibility (CI) pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains and their combinations. Here, we transferred the Wolbachia wMel strain by embryonic microinjection into Aedes albopictus, resulting in the successful establishment of a transinfected line, HM (wAlbAwAlbBwMel), with a triple-strain infection comprising wMel, wAlbA, and wAlbB. Surprisingly, no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but specific removal of wAlbA from the HM (wAlbAwAlbBwMel) line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. The transinfected line showed perfect maternal transmission of the triple infection, with fluctuating egg hatch rates that improved to normal levels after repeated outcrosses with GUA line. Strain-specific qPCR assays showed that wMel and wAlbB were present at the highest densities in the ovaries and midguts, respectively, of the HM (wAlbAwAlbBwMel) mosquitoes. These finding suggest that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup.

Disruption of Neutrophil Extracellular Traps (NETs) Links Mechanical Strain to Post-traumatic Inflammation.

Inflammation after trauma is both critical to normal wound healing and may be highly detrimental when prolonged or unchecked with the potential to impair physiologic healing and promote de novo pathology. Mechanical strain after trauma is associated with impaired wound healing and increased inflammation. The exact mechanisms behind this are not fully elucidated. Neutrophil extracellular traps (NETs), a component of the neutrophil response to trauma, are implicated in a range of pro-inflammatory conditions. In the current study, we evaluated their role in linking movement and inflammation. We found that a link exists between the disruption and amplification of NETs which harbors the potential to regulate the wound's response to mechanical strain, while leaving the initial inflammatory signal necessary for physiologic wound healing intact.

Genes important for survival or reproduction in Varroa destructor identified by RNAi.

The Varroa mite, (Varroa destructor), is the worst threat to honey bee health worldwide. To explore the possibility of using RNA interference to control this pest, we determined the effects of knocking down various genes on Varroa mite survival and reproduction. Double-stranded RNA (dsRNA) of six candidate genes (Da, Pros26S, RpL8, RpL11, RpP0 and RpS13) were synthesized and each injected into Varroa mites, then mite survival and reproduction were assessed. Injection of dsRNA for Da (Daughterless) and Pros26S (Gene for proteasome 26S subunit adenosine triphosphatase) caused a significant reduction in mite survival, with 3.57% ± 1.94% and 30.03% ± 11.43% mites surviving at 72 h post-injection (hpi), respectively. Control mites injected with green fluorescent protein (GFP)-dsRNA showed survival rates of 81.95% ± 5.03% and 82.36 ± 2.81%, respectively. Injections of dsRNA for four other genes (RpL8, RpL11, RpP0 and RpS13) did not affect survival significantly, enabling us to assess their effect on Varroa mite reproduction. The number of female offspring per mite was significantly reduced for mites injected with dsRNA of each of these four genes compared to their GFP-dsRNA controls. Knockdown of the target genes was verified by real-time polymerase chain reaction for two genes important for reproduction (RpL8, RpL11) and one gene important for survival (Pros26S). In conclusion, through RNA interference, we have discovered two genes important for mite survival and four genes important for mite reproduction. These genes could be explored as possible targets for the control of Varroa destructor in the future.

GM-CSF Administration Improves Defects in Innate Immunity and Sepsis Survival in Obese Diabetic Mice.

Sepsis is the leading cause of death in the intensive care unit with an overall mortality rate of 20%. Individuals who are obese and have type 2 diabetes have increased recurrent, chronic, nosocomial infections that worsen the long-term morbidity and mortality from sepsis. Additionally, animal models of sepsis have shown that obese, diabetic mice have lower survival rates compared with nondiabetic mice. Neutrophils are essential for eradication of bacteria, prevention of infectious complications, and sepsis survival. In diabetic states, there is a reduction in neutrophil chemotaxis, phagocytosis, and reactive oxygen species (ROS) generation; however, few studies have investigated the extent to which these deficits compromise infection eradication and mortality. Using a cecal ligation and puncture model of sepsis in lean and in diet-induced obese mice, we demonstrate that obese diabetic mice have decreased "emergency hematopoiesis" after an acute infection. Additionally, both neutrophils and monocytes in obese, diabetic mice have functional defects, with decreased phagocytic ability and a decreased capacity to generate ROS. Neutrophils isolated from obese diabetic mice have decreased transcripts of Axl and Mertk, which partially explains the phagocytic dysfunction. Furthermore, we found that exogenous GM-CSF administration improves sepsis survival through enhanced neutrophil and monocytes phagocytosis and ROS generation abilities in obese, diabetic mice with sepsis.

Obesity and type 2 diabetes mellitus drive immune dysfunction, infection development, and sepsis mortality.

Obesity and type 2 diabetes mellitus (T2D) are global pandemics. Worldwide, the prevalence of obesity has nearly tripled since 1975 and the prevalence of T2D has almost doubled since 1980. Both obesity and T2D are indolent and chronic diseases that develop gradually, with cellular physiologic changes occurring before the clinical signs and symptoms of the diseases become apparent. Individuals with obesity and T2D are physiologically frail and have an increased risk of infections and mortality from sepsis. Improvement in the morbidity and mortality of these at-risk populations would provide a great societal benefit. We believe that the worsened outcomes observed in these patient populations is due to immune system dysfunction that is triggered by the chronic low-grade inflammation present in both diseases. As immune modulatory therapies have been utilized in other chronic inflammatory diseases, there is an emerging role for immune modulatory therapies that target the chronically affected immune pathways in obese and T2D patients. Additionally, bariatric surgery is currently the most successful treatment for obesity and is the only weight loss method that also causes a sustained, substantial improvement of T2D. Consequently, bariatric surgery may also have a role in improving immunity in these patient populations.

Role of complement C5a and histones in septic cardiomyopathy.

Polymicrobial sepsis (after cecal ligation and puncture, CLP) causes robust complement activation with release of C5a. Many adverse events develop thereafter and will be discussed in this review article. Activation of complement system results in generation of C5a which interacts with its receptors (C5aR1, C5aR2). This leads to a series of harmful events, some of which are connected to the cardiomyopathy of sepsis, resulting in defective action potentials in cardiomyocytes (CMs), activation of the NLRP3 inflammasome in CMs and the appearance of extracellular histones, likely arising from activated neutrophils which form neutrophil extracellular traps (NETs). These events are associated with activation of mitogen-activated protein kinases (MAPKs) in CMs. The ensuing release of histones results in defective action potentials in CMs and reduced levels of [Ca2+]i-regulatory enzymes including sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger (NCX) as well as Na+/K+-ATPase in CMs. There is also evidence that CLP causes release of IL-1ß via activation of the NLRP3 inflammasome in CMs of septic hearts or in CMs incubated in vitro with C5a. Many of these events occur after in vivo or in vitro contact of CMs with histones. Together, these data emphasize the role of complement (C5a) and C5a receptors (C5aR1, C5aR2), as well as extracellular histones in events that lead to cardiac dysfunction of sepsis (septic cardiomyopathy).

The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti.

A host's immune system plays a central role in shaping the composition of the microbiota and, in return, resident microbes influence immune responses. Symbiotic associations of the maternally transmitted bacterium Wolbachia occur with a wide range of arthropods. It is, however, absent from the dengue and Zika vector mosquito Aedes aegypti in nature. When Wolbachia is artificially forced to form symbiosis with this new mosquito host, it boosts the basal immune response and enhances the mosquito's resistance to pathogens, including dengue, Zika virus and malaria parasites. The mechanisms involved in establishing a symbiotic relationship between Wolbachia and A. aegypti, and the long-term outcomes of this interaction, are not well understood. Here, we have demonstrated that both the immune deficiency (IMD) and Toll pathways are activated by the Wolbachia strain wAlbB upon its introduction into A. aegypti. Silencing the Toll and IMD pathways via RNA interference reduces the wAlbB load. Notably, wAlbB induces peptidoglycan recognition protein (PGRP)-LE expression in the carcass of A. aegypti, and its silencing results in a reduction of symbiont load. Using transgenic mosquitoes with stage-specific induction of the IMD and Toll pathways, we have shown that elevated wAlbB infection in these mosquitoes is maintained via maternal transmission. These results indicate that host innate immunity is utilized to establish and promote host-microbial symbiosis. Our results will facilitate a long-term projection of the stability of the Wolbachia-A. aegypti mosquito system that is being developed to control dengue and Zika virus transmission to humans.

A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus.

Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes' resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes.

Replacing a native Wolbachia with a novel strain results in an increase in endosymbiont load and resistance to dengue virus in a mosquito vector.

Wolbachia is a maternally transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce pathogen interference and spread into mosquito vector populations makes it possible to develop Wolbachia as a biological control agent for vector-borne disease control. Although Wolbachia induces resistance to dengue virus (DENV), filarial worms, and Plasmodium in mosquitoes, species like Aedes polynesiensis and Aedes albopictus, which carry native Wolbachia infections, are able to transmit dengue and filariasis. In a previous study, the native wPolA in Ae. polynesiensis was replaced with wAlbB from Ae. albopictus, and resulted in the generation of the transinfected "MTB" strain with low susceptibility for filarial worms. In this study, we compare the dynamics of DENV serotype 2 (DENV-2) within the wild type "APM" strain and the MTB strain of Ae. polynesiensis by measuring viral infection in the mosquito whole body, midgut, head, and saliva at different time points post infection. The results show that wAlbB can induce a strong resistance to DENV-2 in the MTB mosquito. Evidence also supports that this resistance is related to a dramatic increase in Wolbachia density in the MTB's somatic tissues, including the midgut and salivary gland. Our results suggests that replacement of a native Wolbachia with a novel infection could serve as a strategy for developing a Wolbachia-based approach to target naturally infected insects for vector-borne disease control.

Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection.

Wolbachia is a maternally transmitted symbiotic bacterium of insects that has been proposed as a potential agent for the control of insect-transmitted diseases. One of the major limitations preventing the development of Wolbachia for malaria control has been the inability to establish inherited infections of Wolbachia in anopheline mosquitoes. Here, we report the establishment of a stable Wolbachia infection in an important malaria vector, Anopheles stephensi. In A. stephensi, Wolbachia strain wAlbB displays both perfect maternal transmission and the ability to induce high levels of cytoplasmic incompatibility. Seeding of naturally uninfected A. stephensi populations with infected females repeatedly resulted in Wolbachia invasion of laboratory mosquito populations. Furthermore, wAlbB conferred resistance in the mosquito to the human malaria parasite Plasmodium falciparum.

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells.

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.

Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti.

Wolbachia are maternally transmitted symbiotic bacteria that can spread within insect populations because of their unique ability to manipulate host reproduction. When introduced to nonnative mosquito hosts, Wolbachia induce resistance to a number of human pathogens, including dengue virus (DENV), Plasmodium, and filarial nematodes, but the molecular mechanism involved is unclear. In this study, we have deciphered how Wolbachia infection affects the Aedes aegypti host in inducing resistance to DENV. The microarray assay indicates that transcripts of genes with functions related to immunity and reduction-oxidation (redox) reactions are up-regulated in Ae. aegypti infected with Wolbachia. Infection with this bacterium leads to induction of oxidative stress and an increased level of reactive oxygen species in its mosquito host. Reactive oxygen species elevation is linked to the activation of the Toll pathway, which is essential in mediating the expression of antioxidants to counterbalance oxidative stress. This immune pathway also is responsible for activation of antimicrobial peptides-defensins and cecropins. We provide evidence that these antimicrobial peptides are involved in inhibition of DENV proliferation in Wolbachia-infected mosquitoes. Utilization of transgenic Ae. aegypti and the RNAi depletion approach has been instrumental in proving the role of defensins and cecropins in the resistance of Wolbachia-infected Ae. aegypti to DENV. These results indicate that a symbiotic bacterium can manipulate the host defense system to facilitate its own persistent infection, resulting in a compromise of the mosquito's ability to host human pathogens. Our discoveries will aid in the development of control strategies for mosquito-transmitted diseases.

Response of the mosquito protein interaction network to dengue infection.

BACKGROUND: Two fifths of the world's population is at risk from dengue. The absence of effective drugs and vaccines leaves vector control as the primary intervention tool. Understanding dengue virus (DENV) host interactions is essential for the development of novel control strategies. The availability of genome sequences for both human and mosquito host greatly facilitates genome-wide studies of DENV-host interactions. RESULTS: We developed the first draft of the mosquito protein interaction network using a computational approach. The weighted network includes 4,214 Aedes aegypti proteins with 10,209 interactions, among which 3,500 proteins are connected into an interconnected scale-free network. We demonstrated the application of this network for the further annotation of mosquito proteins and dissection of pathway crosstalk. Using three datasets based on physical interaction assays, genome-wide RNA interference (RNAi) screens and microarray assays, we identified 714 putative DENV-associated mosquito proteins. An integrated analysis of these proteins in the network highlighted four regions consisting of highly interconnected proteins with closely related functions in each of replication/transcription/translation (RTT), immunity, transport and metabolism. Putative DENV-associated proteins were further selected for validation by RNAi-mediated gene silencing, and dengue viral titer in mosquito midguts was significantly reduced for five out of ten (50.0%) randomly selected genes. CONCLUSIONS: Our results indicate the presence of common host requirements for DENV in mosquitoes and humans. We discuss the significance of our findings for pharmacological intervention and genetic modification of mosquitoes for blocking dengue transmission.

The endosymbiotic bacterium Wolbachia induces resistance to dengue virus in Aedes aegypti.

Genetic strategies that reduce or block pathogen transmission by mosquitoes have been proposed as a means of augmenting current control measures to reduce the growing burden of vector-borne diseases. The endosymbiotic bacterium Wolbachia has long been promoted as a potential vehicle for introducing disease-resistance genes into mosquitoes, thereby making them refractory to the human pathogens they transmit. Given the large overlap in tissue distribution and intracellular localization between Wolbachia and dengue virus in mosquitoes, we conducted experiments to characterize their interactions. Our results show that Wolbachia inhibits viral replication and dissemination in the main dengue vector, Aedes aegypti. Moreover, the virus transmission potential of Wolbachia-infected Ae. aegypti was significantly diminished when compared to wild-type mosquitoes that did not harbor Wolbachia. At 14 days post-infection, Wolbachia completely blocked dengue transmission in at least 37.5% of Ae. aegypti mosquitoes. We also observed that this Wolbachia-mediated viral interference was associated with an elevated basal immunity and increased longevity in the mosquitoes. These results underscore the potential usefulness of Wolbachia-based control strategies for population replacement.

Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection.

Prophenoloxidases (PPOs) are key enzymes of the melanization reaction, which is a prominent defense mechanism of arthropods. The mosquito Aedes aegypti has ten PPO genes in the genome, four of which (PPO1, PPO3, PPO5, and PPO8) were expressed in response to microbial infection. Cactus depletion resulted in transcriptional activation of these four genes, suggesting this up-regulation to be under the control of the Toll pathway. The silencing of Cactus also led to developmental arrest and death of the avian malaria parasite, Plasmodium gallinaceum. We discovered that RUNT-related transcription factor 4 (RUNX4), the orthologue of Drosophila Lozenge, bound to the RUNT binding motif in the promoter of mosquito PPO genes and stimulated the expression of Drosophila PPO-A1 and PPO3 in S2 cell line. The immune effects caused by Cactus depletion were eliminated by double knockdown of Cactus/RUNX4. These findings suggest that RUNX4 regulates PPO gene expression under the control of the Toll pathway and plays a critical role in restricting parasite development.

Characterization of a juvenile hormone-regulated chymotrypsin-like serine protease gene in Aedes aegypti mosquito.

After female mosquitoes ingest blood from vertebrate hosts, exopeptidases and endopeptidases are required for digesting blood proteins in the midgut into amino acids, which female mosquitoes use to build yolk proteins. These proteases are not always present in the midgut, and their diverse expression patterns suggest that production of these enzymes is highly regulated in order to meet specific physiological demands at various stages. Here we report identification of a serine-type protease, JHA15, in the yellow fever mosquito Aedes aegypti. This protein shares high sequence homology with chymotrypsins, and indeed exhibits specific chymotrypsin enzymatic activity. The JHA15 gene is expressed primarily in the midgut of adult female mosquitoes. Our results indicate that its transcription is activated by juvenile hormone in the newly emerged female adults. Although its mRNA profile is similar to that of the early trypsin gene, we found that JHA15 proteins were readily detected in the midgut epithelium cells of both non-blood-fed and blood-fed mosquitoes. Analysis of polysomal RNA further substantiated that synthesis of JHA15 occurs before and shortly after blood feeding. Knocking down expression of JHA15 resulted in no evident phenotypic changes, implying that functional redundancy exists among those proteolytic enzymes.

Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes.

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

A toll receptor and a cytokine, Toll5A and Spz1C, are involved in toll antifungal immune signaling in the mosquito Aedes aegypti.

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

Regulation of lipid metabolism genes, lipid carrier protein lipophorin, and its receptor during immune challenge in the mosquito Aedes aegypti.

In the mosquito Aedes aegypti, the expression of two fat body genes involved in lipid metabolism, a lipid carrier protein lipophorin (Lp) and its lipophorin receptor (LpRfb), was significantly increased after infections with Gram (+) bacteria and fungi, but not with Gram (-) bacteria. The expression of these genes was enhanced after the infection with Plasmodium gallinaceum. RNA interference (RNAi) knockdown of Lp strongly restricted the development of Plasmodium oocysts, reducing their number by 90%. In Vg-DeltaREL1-A transgenic mosquitoes, with gain-of-function phenotype of Toll/REL1 immune pathway activated after blood feeding, both the Lp and LpRfb genes were overexpressed independently of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of Cactus, an IkappaB inhibitor in the Toll/REL1 pathway. These results showed that, in the mosquito fat body, both Lp and LpRfb gene expression were regulated by the Toll/REL1 pathway during immune induction by pathogen and parasite infections. Indeed, the proximal region of the LpRfb promoter contained closely linked binding motifs for GATA and NF-kappaB transcription factors. Transfection and in vivo RNAi knockdown experiments showed that the bindings of both GATA and NF-kappaB transcription factors to the corresponding motif were required for the induction of the LpRfb gene. These findings suggest that lipid metabolism is involved in the mosquito systemic immune responses to pathogens and parasites.