Mast cell-derived IL-10 protects intestinal barrier integrity during malaria in mice and regulates parasite transmission to Anopheles stephensi with a female-biased immune response.

Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.

Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti.

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.

Starving the Beast: Limiting Coenzyme A Biosynthesis to Prevent Disease and Transmission in Malaria.

Malaria parasites must acquire all necessary nutrients from the vertebrate and mosquito hosts to successfully complete their life cycle. Failure to acquire these nutrients can limit or even block parasite development and presents a novel target for malaria control. One such essential nutrient is pantothenate, also known as vitamin B5, which the parasite cannot synthesize de novo and is required for the synthesis of coenzyme A (CoA) in the parasite. This review examines pantothenate and the CoA biosynthesis pathway in the human-mosquito-malaria parasite triad and explores possible approaches to leverage the CoA biosynthesis pathway to limit malaria parasite development in both human and mosquito hosts. This includes a discussion of sources for pantothenate for the mosquito, human, and parasite, examining the diverse strategies used by the parasite to acquire substrates for CoA synthesis across life stages and host resource pools and a discussion of drugs and alternative approaches being studied to disrupt CoA biosynthesis in the parasite. The latter includes antimalarial pantothenate analogs, known as pantothenamides, that have been developed to target this pathway during the human erythrocytic stages. In addition to these parasite-targeted drugs, we review studies of mosquito-targeted allosteric enzymatic regulators known as pantazines as an approach to limit pantothenate availability in the mosquito and subsequently deprive the parasite of this essential nutrient.

A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power.

BACKGROUND: Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. "Next generation" high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose. RESULTS: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807-819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123-139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic. CONCLUSIONS: The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

Nonlethal Plasmodium yoelii Infection Drives Complex Patterns of Th2-Type Host Immunity and Mast Cell-Dependent Bacteremia.

Malaria strongly predisposes to bacteremia, which is associated with sequestration of parasitized red blood cells and increased gastrointestinal permeability. The mechanisms underlying this disruption are poorly understood. Here, we evaluated the expression of factors associated with mast cell activation and malaria-associated bacteremia in a rodent model. C57BL/6J mice were infected with Plasmodium yoeliiyoelli 17XNL, and blood and tissues were collected over time to assay for circulating levels of bacterial 16S DNA, IgE, mast cell protease 1 (Mcpt-1) and Mcpt-4, Th1 and Th2 cytokines, and patterns of ileal mastocytosis and intestinal permeability. The anti-inflammatory cytokines (interleukin-4 [IL-4], IL-6, and IL-10) and MCP-1/CCL2 were detected early after P. yoeliiyoelii 17XNL infection. This was followed by the appearance of IL-9 and IL-13, cytokines known for their roles in mast cell activation and growth-enhancing activity as well as IgE production. Later increases in circulating IgE, which can induce mast cell degranulation, as well as Mcpt-1 and Mcpt-4, were observed concurrently with bacteremia and increased intestinal permeability. These results suggest that P. yoeliiyoelii 17XNL infection induces the production of early cytokines that activate mast cells and drive IgE production, followed by elevated IgE, IL-9, and IL-13 that maintain and enhance mast cell activation while disrupting the protease/antiprotease balance in the intestine, contributing to epithelial damage and increased permeability.

Inhibition of JNK signaling in the Asian malaria vector Anopheles stephensi extends mosquito longevity and improves resistance to Plasmodium falciparum infection.

Malaria is a global health concern caused by infection with Plasmodium parasites. With rising insecticide and drug resistance, there is a critical need to develop novel control strategies, including strategies to block parasite sporogony in key mosquito vector species. MAPK signaling pathways regulated by extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs) c-Jun N-terminal kinases (JNKs) and p38 MAPKs are highly conserved across eukaryotes, including mosquito vectors of the human malaria parasite Plasmodium falciparum. Some of these pathways in mosquitoes have been investigated in detail, but the mechanisms of integration of parasite development and mosquito fitness by JNK signaling have not been elucidated. To this end, we engineered midgut-specific overexpression of MAPK phosphatase 4 (MKP4), which targets the SAPKs, and used two potent and specific JNK small molecule inhibitors (SMIs) to assess the effects of JNK signaling manipulations on Anopheles stephensi fecundity, lifespan, intermediary metabolism, and P. falciparum development. MKP4 overexpression and SMI treatment reduced the proportion of P. falciparum-infected mosquitoes and decreased oocyst loads relative to controls. SMI-treated mosquitoes exhibited no difference in lifespan compared to controls, whereas genetically manipulated mosquitoes exhibited extended longevity. Metabolomics analyses of SMI-treated mosquitoes revealed insights into putative resistance mechanisms and the physiology behind lifespan extension, suggesting for the first time that P. falciparum-induced JNK signaling reduces mosquito longevity and increases susceptibility to infection, in contrast to previously published reports, likely via a critical interplay between the invertebrate host and parasite for nutrients that play essential roles during sporogonic development.

Two insulin-like peptides differentially regulate malaria parasite infection in the mosquito through effects on intermediary metabolism.

Insulin-like peptides (ILPs) play important roles in growth and metabolic homeostasis, but have also emerged as key regulators of stress responses and immunity in a variety of vertebrates and invertebrates. Furthermore, a growing literature suggests that insulin signaling-dependent metabolic provisioning can influence host responses to infection and affect infection outcomes. In line with these studies, we previously showed that knockdown of either of two closely related, infection-induced ILPs, ILP3 and ILP4, in the mosquito Anopheles stephensi decreased infection with the human malaria parasite Plasmodium falciparum through kinetically distinct effects on parasite death. However, the precise mechanisms by which ILP3 and ILP4 control the response to infection remained unknown. To address this knowledge gap, we used a complementary approach of direct ILP supplementation into the blood meal to further define ILP-specific effects on mosquito biology and parasite infection. Notably, we observed that feeding resulted in differential effects of ILP3 and ILP4 on blood-feeding behavior and P. falciparum development. These effects depended on ILP-specific regulation of intermediary metabolism in the mosquito midgut, suggesting a major contribution of ILP-dependent metabolic shifts to the regulation of infection resistance and parasite transmission. Accordingly, our data implicate endogenous ILP signaling in balancing intermediary metabolism for the host response to infection, affirming this emerging tenet in host-pathogen interactions with novel insights from a system of significant public health importance.

Human IGF1 regulates midgut oxidative stress and epithelial homeostasis to balance lifespan and Plasmodium falciparum resistance in Anopheles stephensi.

Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013-0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases.

Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.

Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.

Enhanced transmission of malaria parasites to mosquitoes in a murine model of type 2 diabetes.

BACKGROUND: More than half of the world's population is at risk of malaria and simultaneously, many malaria-endemic regions are facing dramatic increases in the prevalence of type 2 diabetes. Studies in murine malaria models have examined the impact of malaria infection on type 2 diabetes pathology, it remains unclear how this chronic metabolic disorder impacts the transmission of malaria. In this report, the ability type 2 diabetic rodents infected with malaria to transmit parasites to Anopheles stephensi mosquitoes is quantified. METHODS: The infection prevalence and intensity of An. stephensi mosquitoes that fed upon control or type 2 diabetic C57BL/6 db/db mice infected with either lethal Plasmodium berghei NK65 or non-lethal Plasmodium yoelii 17XNL murine malaria strains were determined. Daily parasitaemias were also recorded. RESULTS: A higher percentage of mosquitoes (87.5 vs 61.5 % for P. yoelii and 76.9 vs 50 % for P. berghei) became infected following blood feeding on Plasmodium-infected type 2 diabetic mice compared to mosquitoes that fed on infected control animals, despite no significant differences in circulating gametocyte levels. CONCLUSIONS: These results suggest that type 2 diabetic mice infected with malaria are more efficient at infecting mosquitoes, raising the question of whether a similar synergy exists in humans.

Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

Quantitative phase imaging by gradient retardance optical microscopy.

Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of wavefront distortion and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few QPI applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, we introduce Gradient Retardance Optical Microscopy (GROM) for QPI of both thin and thick samples. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a QPI platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. We successfully tested GROM on a diverse range of specimens, from microbes and red blood cells to optically thick (~ 300 µm) plant roots without fixation or clearing.

Community-serving research addressing climate change impacts on vector-borne diseases.

The impacts of climate change on vector-borne diseases are uneven across human populations. This pattern reflects the effect of changing environments on the biology of transmission, which is also modulated by social and other inequities. These disparities are also linked to research outcomes that could be translated into tools for transmission reduction, but are not necessarily actionable in the communities where transmission occurs. The transmission of vector-borne diseases could be averted by developing research that is both hypothesis-driven and community-serving for populations affected by climate change, where local communities interact as equal partners with scientists, developing and implementing research projects with the aim of improving community health. In this Personal View, we share five principles that have guided our research practice to serve the needs of communities affected by vector-borne diseases.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1ß (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia.

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing L-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans.

Human IGF1 extends lifespan and enhances resistance to Plasmodium falciparum infection in the malaria vector Anopheles stephensi.

The highly conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway regulates metabolism, development, lifespan and immunity across a wide range of organisms. Previous studies have shown that human insulin ingested in the blood meal can activate mosquito IIS, resulting in attenuated lifespan and increased malaria parasite infection. Because human IGF1 is present at higher concentrations in blood than insulin and is functionally linked with lifespan and immune processes, we predicted that human IGF1 ingested in a blood meal would affect lifespan and malaria parasite infection in the mosquito Anopheles stephensi. Here we demonstrate that physiological levels of ingested IGF1, like insulin, can persist intact in the blood-filled midgut for up to 30 h and disseminate into the mosquito body, and that both peptides activate IIS in mosquito cells and midgut. At these same levels, ingested IGF1 alone extended average mosquito lifespan by 23% compared with controls and, more significantly, when ingested in infected blood meals, reduced the prevalence of Plasmodium falciparum-infected mosquitoes by >20% and parasite load by 35-50% compared with controls. Thus, the effects of ingested IGF1 on mosquito lifespan and immunity are opposite to those of ingested insulin. These results offer the first evidence that insect cells can functionally discriminate between mammalian insulin and IGF1. Further, in light of previous success in genetically targeting IIS to alter mosquito lifespan and malaria parasite transmission, this study indicates that a more complete understanding of the IIS-activating ligands in blood can be used to optimize transgenic strategies for malaria control.

In vivo, in vitro, and in silico studies suggest a conserved immune module that regulates malaria parasite transmission from mammals to mosquitoes.

Human malaria can be caused by the parasite Plasmodium falciparum that is transmitted by female Anopheles mosquitoes. "Immunological crosstalk" between the mammalian and anopheline hosts for Plasmodium functions to control parasite numbers. Key to this process is the mammalian cytokine transforming growth factor-ß1 (TGF-ß1). In mammals, TGF-ß1 regulates inducible nitric oxide (NO) synthase (iNOS) both positively and negatively. In some settings, high levels of NO activate latent TGF-ß1, which in turn suppresses iNOS expression. In the mosquito, ingested TGF-ß1 induces A. stephensi NOS (AsNOS), which limits parasite development and which in turn is suppressed by activation of the mosquito homolog of the mitogen-activated protein kinases MEK and ERK. Computational models linking TGF-ß1, AsNOS, and MEK/ERK were developed to provide insights into this complex biology. An initial Boolean model suggested that, as occurs in mammalian cells, MEK/ERK and AsNOS would oscillate upon ingestion of TGF-ß1. An ordinary differential equation (ODE) model further supported the hypothesis of TGF-ß1-induced multiphasic behavior of MEK/ERK and AsNOS. To achieve this multiphasic behavior, the ODE model was predicated on the presence of constant levels of TGF-ß1 in the mosquito midgut. Ingested TGF-ß1, however, did not exhibit this behavior. Accordingly, we hypothesized and experimentally verified that ingested TGF-ß1 induces the expression of the endogenous mosquito TGF-ß superfamily ligand As60A. Computational simulation of these complex, cross-species interactions suggested that TGF-ß1 and NO-mediated induction of As60A expression together may act to maintain multiphasic AsNOS expression via MEK/ERK-dependent signaling. We hypothesize that multiphasic behavior as represented in this model allows the mosquito to balance the conflicting demands of parasite killing and metabolic homeostasis in the face of damaging inflammation.

Longitudinal and Cross-sectional Analyses of Asymptomatic HIV-1/Malaria Co-infection in Kisumu County, Kenya.

Individuals infected with HIV-1 experience more frequent and more severe episodes of malaria and are likely to harbor asymptomatic parasitemia, thus potentially making them more efficient reservoirs of malaria. Two studies (cross-sectional and longitudinal) were designed in sequence between 2015-2018 and 2018-2020, respectively, to test the hypothesis that HIV-1 infected individuals have higher prevalence of asymptomatic parasitemia and gametocytemia than the HIV-1 negatives. This article describes the overall design of the two studies, encompassing data for the longitudinal study and additional data to the previously published baseline data for the cross-sectional study. In the cross-sectional study, HIV-1 positive participants were significantly older, more likely to be male, and more likely to have parasitemia relative to HIV-1 negatives (P < 0.01). In the longitudinal study, 300 participants were followed for 6 months. Of these, 102 were HIV-1 negative, 106 were newly diagnosed HIV-1 positive, and 92 were HIV-1 positive and on antiretroviral therapy, including antifolates, at enrollment. Overall parasitemia positivity at enrollment was 17.3% (52/300). Of these, 44% (23/52) were HIV-1 negative, 52% (27/52) were newly diagnosed HIV-1 positives, and only 4% (2/52) were HIV-1 positive and on treatment. Parasitemia for those on stable antiretroviral therapy was significantly lower (hazard ratio: 0.51, P < 0.001), compared with the HIV-1-negatives. On follow-up, there was a significant decline in parasitemia prevalence (hazard ratio: 0.74, P < 0.001) among the HIV patients newly initiated on antiretroviral therapy including trimethoprim-sulfamethoxasole. These data highlight the impact of HIV-1 and HIV treatment on asymptomatic parasitemia over time.

Ingested histamine and serotonin interact to alter Anopheles stephensi feeding and flight behavior and infection with Plasmodium parasites.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.

Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum.

We showed previously that ingested human insulin activates the insulin/IGF-1 signaling pathway in Anopheles stephensi and increases the susceptibility of these mosquitoes to Plasmodium falciparum. In other organisms, insulin can alter immune responsiveness through regulation of NF-κB transcription factors, critical elements for innate immunity that are also central to mosquito immunity. We show here that insulin signaling decreased expression of NF-κB-regulated immune genes in mosquito cells stimulated with either bacterial or malarial soluble products. Further, human insulin suppressed mosquito immunity through sustained phosphatidylinositol 3-kinase activation, since inhibition of this pathway led to decreased parasite development in the mosquito. Together, these data demonstrate that activation of the insulin/IGF-1 signaling pathway by ingested human insulin can alter NF-κB-dependent immunity, and ultimately the susceptibility, of mosquitoes to P. falciparum.