Influenza enhances host susceptibility to non-pulmonary invasive Streptococcus pyogenes infections.

Streptococcus pyogenes (group A streptococcus; GAS) causes a variety of invasive diseases (iGAS) such as bacteremia, toxic shock syndrome, and pneumonia, which are associated with high mortality despite the susceptibility of the bacteria to penicillin ex vivo. Epidemiologic studies indicate that respiratory influenza virus infection is associated with an increase in the frequency of iGAS diseases, including those not directly involving the lung. We modified a murine model of influenza A (IAV)-GAS superinfection to determine if viral pneumonia increased the susceptibility of mice subsequently infected with GAS in the peritoneum. The results showed that respiratory IAV infection increased the morbidity (weight loss) of mice infected intraperitoneally (i.p.) with GAS 3, 5, and 10 d after the initial viral infection. Mortality was also significantly increased when mice were infected with GAS 3 and 5 d after pulmonary IAV infection. Increased mortality among mice infected with virus 5 d prior to bacterial infection correlated with increased dissemination of GAS from the peritoneum to the blood, spleen, and lungs. The interval was also associated with a significant increase in the pro-inflammatory cytokines IFN-γ, IL-12, TNF-α, MCP-1 and IL-27 in sera. We conclude, using a murine model, that respiratory influenza virus infection increases the likelihood and severity of systemic iGAS disease, even when GAS infection does not originate in the respiratory tract.

UBXN2A suppresses the Rictor-mTORC2 signaling pathway, an established tumorigenic pathway in human colorectal cancer.

The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.

Enhancing Anti-Tumorigenic Efficacy of Eugenol in Human Colon Cancer Cells Using Enzyme-Responsive Nanoparticles.

This study is focused on the selective delivery and release of the plant-based anticancer compound eugenol (EUG) in colorectal cancer cells (CRC). EUG is an apoptotic and anti-growth compound in diverse malignant tumors, including CRC. However, EUG's rapid metabolization, excretion, and side effects on normal cells at higher dosages are major limitations of its therapeutic potential. To address this problem, we developed a "smart" enzyme-responsive nanoparticle (eNP) loaded with EUG that exposes tumors to a high level of the drug while keeping its concentration low among healthy cells. We demonstrated that EUG induces apoptosis in CRC cells irrespective of their grades in a dose- and time-dependent manner. EUG significantly decreases cancer cell migration, invasion, and the population of colon cancer stem cells, which are key players in tumor metastasis and drug resistance. The "smart" eNPs-EUG show a high affinity to cancer cells with rapid internalization with no affinity toward normal colon epithelial cells. NPs-EUG enhanced the therapeutic efficacy of EUG measured by a cell viability assay and showed no toxicity effect on normal cells. The development of eNPs-EUG is a promising strategy for innovative anti-metastatic therapeutics.

Examination of vertical transmission of Bartonella quintana in body lice following multiple infectious blood meals.

Bartonella quintana is a re-emerging louse-borne pathogen. Horizontal transmission from the body louse vector (Pediculus humanus humanus) to a human host occurs through contact with infectious louse feces containing a high concentration of the bacteria. However, questions have remained about whether vertical transmission from infected vectors to their progeny, which could significantly influence the dynamics of transmission to humans, occurs in body lice. To address this subject, we performed a series of controlled laboratory experiments that examined the presence of B. quintana on the surface of and within eggs produced by female body lice that were provisioned multiple infectious blood meals to recapitulate the natural pathogen acquisition process. Our results demonstrate that B. quintana DNA can be detected from the surface of eggs by qPCR due to vertical transfer of infectious feces to the egg sheath during or after oviposition. However, viable B. quintana could not be cultured from the hemolymph of adult female lice or from within eggs that were surface sterilized, indicating a lack of true transovarial transmission. Based on this evidence, vertical transfer of B. quintana from infected adult lice to their eggs probably has a limited impact on the dynamics of transmission to humans.

A Comparative Study of Body Lice and Bed Bugs Reveals Factors Potentially Involved in Differential Vector Competence for the Relapsing Fever Spirochete Borrelia recurrentis.

Borrelia recurrentis is the causative agent of louse-borne relapsing fever and the only Borrelia species transmitted by an insect rather than a tick vector. While bed bugs (Cimex lectularius L.) are not established vectors of any human pathogens, a recent study reported that they may be competent vectors of B. recurrentis. However, many aspects of infection and transmission remain unclear in this possible secondary vector. Here, we carried out several quantitative laboratory studies to gain a better understanding of the host suitability of bed bugs relative to the established body louse vector as well as the factors that may affect the ability of bed bugs to transmit the pathogen. We fed bed bugs B. recurrentis and estimated the level and duration of infection in the hemolymph using live imaging. We performed quantitative PCR (qPCR) to examine whole-body spirochete levels and the occurrence of vertical transmission to progeny. We also developed an assay to compare the amounts of force required to release infectious hemolymph from recently engorged bed bugs and body lice. Finally, we analyzed humoral antibacterial activity in the hemolymph, hemolymph pH, and hemocyte activity in both insect species. Our results confirm that within 24 h of ingestion, B. recurrentis can penetrate the midgut epithelium of bed bugs and enter the hemolymph, overcoming a major host barrier, as in body lice. Once in the hemolymph, spirochetes remain visible for at least 4 days. Moreover, we show that bed bugs are more physically susceptible to crushing than body lice, suggesting that crushing is a feasible route for the natural dissemination of B. recurrentis from the hemolymph of bed bugs, as for body lice. Nonetheless, our data also indicate that bed bugs are suboptimal hosts for B. recurrentis, as the bacterium does not appear to proliferate to high levels or stably colonize the hemolymph and exhibits pleomorphism in this environment. In particular, our data suggest that hemolymph pH and unique cellular immune responses, rather than humoral effectors, may be involved in limiting spirochete survival in bed bugs. Notably, we document the formation of extracellular DNA traps by bed bug hemocytes for the first time. For these reasons, while bed bugs may be capable of limited transmission given their ecology, vector competence is probably minimal relative to body lice. Additional mechanistic studies of human pathogen infection of bed bugs may provide much-needed insight into the biological factors that restrict their ability to act as vectors and may reveal novel mechanisms of immunity.

Pre-clinical safety and therapeutic efficacy of a plant-based alkaloid in a human colon cancer xenograft model.

A high-throughput drug screen revealed that veratridine (VTD), a natural plant alkaloid, induces expression of the anti-cancer protein UBXN2A in colon cancer cells. UBXN2A suppresses mortalin, a heat shock protein, with dominant roles in cancer development including epithelial-mesenchymal transition (EMT), cancer cell stemness, drug resistance, and apoptosis. VTD-dependent expression of UBXN2A leads to the deactivation of mortalin in colon cancer cells, making VTD a potential targeted therapy in malignant tumors with high levels of mortalin. VTD was used clinically for the treatment of hypertension in decades past. However, the discovery of newer antihypertensive drugs and concerns over potential neuro- and cardiotoxicity ended the use of VTD for this purpose. The current study aims to determine the safety and efficacy of VTD at doses sufficient to induce UBXN2A expression in a mouse model. A set of flow-cytometry experiments confirmed that VTD induces both early and late apoptosis in a dose-dependent manner. In vivo intraperitoneal (IP) administration of VTD at 0.1 mg/kg every other day (QOD) for 4 weeks effectively induced expression of UBXN2A in the small and large intestines of mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays on tissues collected from VTD-treated animals demonstrated VTD concentrations in the low pg/mg range. To address concerns regarding neuro- and cardiotoxicity, a comprehensive set of behavioral and cardiovascular assessments performed on C57BL/6NHsd mice revealed that VTD generates no detectable neurotoxicity or cardiotoxicity in animals receiving 0.1 mg/kg VTD QOD for 30 days. Finally, mouse xenograft experiments in athymic nude mice showed that VTD can suppress tumor growth. The main causes for the failure of experimental oncologic drug candidates are lack of sufficient safety and efficacy. The results achieved in this study support the potential utility of VTD as a safe and efficacious anti-cancer molecule.

A Francisella tularensis-Like Bacterium in Tropical Bed Bugs from Madagascar.

The genus Francisella includes several highly virulent human pathogens and some tick endosymbionts. Francisella infections are acquired by humans through contact with vertebrate animal reservoirs or contaminated water or dust. The species Francisella tularensis can also be transmitted by arthropods including ticks, mosquitoes, and flies. For the first time, we describe the molecular detection of an F. tularensis-like bacterium in bed bugs from samples collected in rural Madagascar. This finding suggests a potential involvement of bed bugs in the ecology of Francisella. The role of bed bugs as possible hosts, reservoirs, or vectors of Francisella spp. should be further investigated.

Targeting Colon Cancer Cells with Enzyme-Triggered Casein-Gated Release of Cargo from Mesoporous Silica-Based Nanoparticles.

Colorectal cancer (CRC) is one of the most widely diagnosed cancers worldwide. Despite notable improvements in therapeutic strategies available to CRC patients, late stages of CRC have a higher incidence rate of drug resistance, which is associated with a higher mortality rate. The development of therapeutic strategies that use nanoparticles as a drug delivery system has become one of the most promising potential approaches for cancer therapy. Previous studies have shown that a natural plant alkaloid, veratridine (VTD), suppresses colon cancer cell migration and invasion, two essential factors in tumor metastasis, through activation of the gene that encodes the tumor-suppressor protein UBXN2A. The goal of this study is to develop a nanoassembly to selectively deliver VTD to cancer cells and release it on demand while leaving normal cells intact. We packaged the targeted therapy anticancer molecule VTD inside mesoporous silica nanoparticles (MSNs) impermeable to the blood-brain barrier (BBB) and with selective affinity to CRC cells and sealed the VTD-loaded nanoparticles with an enzymatically cleavable protein. The particles will deliver and release VTD only at the targeted colorectal tumor sites. Since the enzyme MMP-7 protease is dominantly secreted by CRC cells, the release triggered by the enzymes will increase VTD concentration at tumor cells, enhancing the efficiency of the new therapy. We have proven the selective affinity of two types of VTD-carrying particles to CRC cells and enzyme- or acid-triggered VTD release. Negatively surface-charged MSNs showed significant affinity toward positively charged cancer cells but not negatively charged normal fibroblast colon cells, making VTD-MSNs a promising anticancer drug with minimal side effects.

Effects of NF-kB Signaling Inhibitors on Bed Bug Resistance to Orally Provisioned Entomopathogenic Bacteria.

Bed bugs are globally important pests and there is an ongoing need for the development and improvement of bed bug control tools. Though promising against other insect pests, the exploration of biological methods for bed bug control is limited. Previously, we identified several species of bacteria that have entomopathogenic effects against bed bugs when ingested. We also described the conservation of several antibacterial responses in bed bugs, including the expression of immune effector genes regulated by NF-kB transcription factors through the Toll and immune deficiency (IMD) signaling pathways. Accordingly, we predicted that chemical inhibition of NF-kB signaling could reduce bed bug resistance to orally provisioned entomopathogenic bacteria, potentially improving their effectiveness as biological control agents. In the present study, we administered four small molecule inhibitors of NF-kB signaling (BMS345541, IKK16, IMD0354, Takinib) to bed bugs by feeding them in a blood meal. We then quantified basal mortality and mortality in response to oral infection with two different entomopathogenic bacteria (Pseudomonas entomophila and Bacillus thuringiensis israelensis). None of the NF-kB signaling inhibitors tested increased mortality above control levels when administered alone, suggesting a lack of direct toxicity. However, one inhibitor (IKK16) significantly enhanced the rate of mortality from oral infection with P. entomophila. Enhanced mortality was independent of direct effects of IKK16 on P. entomophila growth in vitro but was associated with higher bacterial loads in vivo (i.e., reduced resistance). Together, these results provide new insight into the regulation of the bed bug immune system and suggest that administration of entomopathogens in combination with inhibition of immune signaling pathways to reduce infection resistance may be effective for biological control of bed bugs.

Molecular analysis of the blood meals and bacterial communities of bed bugs (Cimex lectularius L.) to assess interactions with alternative hosts.

Common bed bugs (Cimex lectularius L.) are hematophagous pests present in urban environments across the globe. It is widely established that they have a strong host preference for humans. However, there are records of C. lectularius feeding upon a range of mammalian and avian hosts, including rodents, in the field. There is little information available about how frequently common bed bugs feed on alternative hosts in residential settings, but understanding this phenomenon has implications for both management of infestations and public health. Here, we examined cohorts of C. lectularius collected from 13 different dwellings in the state of New Jersey, USA, that were known to be simultaneously infested with house mice (Mus musculus domesticus). Host-specific quantitative polymerase chain reaction (qPCR) was used to determine if blood meals were taken from mice, while 16S rRNA gene amplicon sequencing was used to screen the bed bugs for the presence of zoonotic bacterial pathogens. We found no evidence that any of the bed bugs we collected fed on mice. Furthermore, the insects harbored depauperate bacterial communities that did not include known human pathogens. However, host-specific qPCR detected feline DNA in a pool of bed bugs from one dwelling, suggesting that interaction with domestic pets should be further investigated. Although sampling in this study was limited, the approach described herein will be useful for additional studies of the interactions between bed bugs and alternative blood meal hosts.

Establishment of Amblyomma americanum populations and new records of Borrelia burgdorferi-infected Ixodes scapularis in South Dakota.

Tick-borne diseases are an emerging public health threat in the United States, but surveillance is lacking in some regions. To advance current knowledge of the ecology of ticks and tick-borne diseases in South Dakota, we conducted a survey in the summer of 2019, focusing on the eastern counties of the state. We collected and identified 266 ticks and a subset were tested for the presence of Borrelia burgdorferi by polymerase chain reaction (PCR). Dermacentor variabilis, a ubiquitous species in the state, was the most commonly identified tick, present in all counties surveyed. However, we also identified 15 Amblyomma americanum from three different locations, providing the first evidence of established populations in the state and expanding the range of this species. In addition, we identified 22 Ixodes scapularis from five different locations, confirming a previous report of an established population in the state. Two adult I. scapularis from two different sites were found to harbor B. burgdorferi, including an individual from Lincoln County, suggesting the ongoing presence of the pathogen in tick populations in the state and representing its southwestern-most detection in the midwest United States. These findings provide important information for assessing and monitoring the public health risk from tick-borne diseases in an area where surveillance is lacking.

The Persistence of Escherichia coli Infection in German Cockroaches (Blattodea: Blattellidae) Varies Between Host Developmental Stages and is Influenced by the Gut Microbiota.

The German cockroach, Blatella germanica (L.), is a suspected vector of several enteric bacterial pathogens, including Escherichia coli, among livestock and humans. However, little is known about the factors that influence bacterial transmission by cockroaches. Here, we orally infected B. germanica with various laboratory and field strains of E. coli and examined bacterial titers over time to shed new light on the factors that influence the dynamics of infection. Our results reveal that a laboratory strain of E. coli is largely cleared within 48 h while one field isolate can persist in a majority of cockroaches (80-100%) for longer than 3 d with minimal impact on cockroach longevity. We also find that the ability to clear some strains of E. coli is greater in cockroach nymphs than adults. Notably, no differential expression of the antimicrobial gene lysozyme was observed between nymphs and adults or in infected groups. However, clearance of E. coli was significantly reduced in gnotobiotic cockroaches that were reared in the absence of environmental bacteria, suggesting a protective role for the microbiota against exogenous bacterial pathogens. Together, these results demonstrate that the interactions between cockroaches and enteric bacterial pathogens are highly dynamic and influenced by a combination of microbial, host, and environmental parameters. Such factors may affect the disease transmission capacity of cockroaches in nature and should be further considered in both lab and field studies.

Ex vivo characterization of the circulating hemocytes of bed bugs and their responses to bacterial exposure.

Bed bugs (Cimex spp.) are urban pests of global importance. Knowledge of the immune system of bed bugs has implications for understanding their susceptibility to biological control agents, their potential to transmit human pathogens, and the basic comparative immunology of insects. Nonetheless, the immunological repertoire of the family Cimicidae remains poorly characterized. Here, we use microscopy, flow cytometry, and RNA sequencing to provide a basal characterization of the circulating hemocytes of the common bed bug, Cimex lectularius. We also examine the responses of these specialized cells to E. coli exposure using the same techniques. Our results show that circulating hemocytes are comprised of at least four morphologically distinct cell types that are capable of phagocytosis, undergo degranulation, and exhibit additional markers of activation following stimulation, including size shift and DNA replication. Furthermore, transcriptomic profiling reveals expression of predicted Toll/IMD signaling pathway components, antimicrobial effectors and other potentially immunoresponsive genes in these cells. Together, our data demonstrate the conservation of several canonical cellular immune responses in the common bed bug and provide a foundation for additional mechanistic immunological studies with specific pathogens of interest.

Microbiome Differences Between Human Head and Body Lice Ecotypes Revealed by 16S RRNA Gene Amplicon Sequencing.

Human head lice and body lice (Pediculus humanus) are neglected ectoparasites. Head lice continue to be prevalent in children worldwide, and insecticide resistance in these insects has complicated their treatment. Meanwhile, body lice, which are most common in the developing world, are resurging among marginalized populations in developed nations. Today, the microbiome is being increasingly recognized as a key mediator of insect physiology. However, the microbial communities that inhabit human lice have remained unknown beyond only a few species of bacteria. Knowledge of the microbiomes of head and body lice could improve our understanding of the observed physiological differences between the 2 ecotypes and potentially inform the development of novel interventions against lice infestations and louse-borne infectious diseases. Toward these goals, here we performed 16S rRNA gene amplicon sequencing to characterize the microbiomes of both head and body lice and identify patterns of interest among these communities. Our data reveal that head and body lice harbor limited but distinct communities of bacteria that include known intracellular endosymbionts ("Candidatus Riesia pediculicola"), extracellular bacteria that may be horizontally acquired from the host environment, and a number of taxa of known or potential public health significance. Notably, in body lice, the relative abundance of vertically transmitted endosymbionts is lower than in head lice, which is a significant driver of greater alpha diversity. Further, several differentially abundant non-endosymbiont taxa and differences in beta diversity were observed between head lice and body lice. These findings support the hypothesis that microbiome differences could contribute to the divergence between human louse ecotypes and underscore the need for future studies to better comprehend the acquisition and physiological roles of human lice microbiomes.

Integrin αvß8-expressing tumor cells evade host immunity by regulating TGF-ß activation in immune cells.

TGF-ß is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-ß (L-TGF-ß) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-ß. Binding of L-TGF-ß to integrin αvß8 results in activation of TGF-ß. We engineered and used αvß8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect ß8 in human tumors. Inhibition of αvß8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. ß8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-ß, suggesting that tumor cell αvß8 serves as a platform for activating cell-surface L-TGF-ß presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to ß8 inhibition with major increases in chemokine and tumor-eliminating genes. High ß8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvß8 is a PD-1/PD-L1-independent immunotherapeutic target.

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.

Mast cells and histamine alter intestinal permeability during malaria parasite infection.

Co-infections with malaria and non-typhoidal Salmonella serotypes (NTS) can present as life-threatening bacteremia, in contrast to self-resolving NTS diarrhea in healthy individuals. In previous work with our mouse model of malaria/NTS co-infection, we showed increased gut mastocytosis and increased ileal and plasma histamine levels that were temporally associated with increased gut permeability and bacterial translocation. Here, we report that gut mastocytosis and elevated plasma histamine are also associated with malaria in an animal model of falciparum malaria, suggesting a broader host distribution of this biology. In support of mast cell function in this phenotype, malaria/NTS co-infection in mast cell-deficient mice was associated with a reduction in gut permeability and bacteremia. Further, antihistamine treatment reduced bacterial translocation and gut permeability in mice with malaria, suggesting a contribution of mast cell-derived histamine to GI pathology and enhanced risk of bacteremia during malaria/NTS co-infection.

Inflammation-associated alterations to the intestinal microbiota reduce colonization resistance against non-typhoidal Salmonella during concurrent malaria parasite infection.

Childhood malaria is a risk factor for disseminated infections with non-typhoidal Salmonella (NTS) in sub-Saharan Africa. While hemolytic anemia and an altered cytokine environment have been implicated in increased susceptibility to NTS, it is not known whether malaria affects resistance to intestinal colonization with NTS. To address this question, we utilized a murine model of co-infection. Infection of mice with Plasmodium yoelii elicited infiltration of inflammatory macrophages and T cells into the intestinal mucosa and increased expression of inflammatory cytokines. These mucosal responses were also observed in germ-free mice, showing that they are independent of the resident microbiota. Remarkably, P. yoelii infection reduced colonization resistance of mice against S. enterica serotype Typhimurium. Further, 16S rRNA sequence analysis of the intestinal microbiota revealed marked changes in the community structure. Shifts in the microbiota increased susceptibility to intestinal colonization by S. Typhimurium, as demonstrated by microbiota reconstitution of germ-free mice. These results show that P. yoelii infection, via alterations to the microbial community in the intestine, decreases resistance to intestinal colonization with NTS. Further they raise the possibility that decreased colonization resistance may synergize with effects of malaria on systemic immunity to increase susceptibility to disseminated NTS infections.

Plasmodium falciparum suppresses the host immune response by inducing the synthesis of insulin-like peptides (ILPs) in the mosquito Anopheles stephensi.

The insulin-like peptides (ILPs) and their respective signaling and regulatory pathways are highly conserved across phyla. In invertebrates, ILPs regulate diverse physiological processes, including metabolism, reproduction, behavior, and immunity. We previously reported that blood feeding alone induced minimal changes in ILP expression in Anopheles stephensi. However, ingestion of a blood meal containing human insulin or Plasmodium falciparum, which can mimic insulin signaling, leads to significant increases in ILP expression in the head and midgut, suggesting a potential role for AsILPs in the regulation of P. falciparum sporogonic development. Here, we show that soluble P. falciparum products, but not LPS or zymosan, directly induced AsILP expression in immortalized A. stephensi cells in vitro. Further, AsILP expression is dependent on signaling by the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositol 3'-kinase (PI3K)/Akt branches of the insulin/insulin-like growth factor signaling (IIS) pathway. Inhibition of P. falciparum-induced ILPs in vivo decreased parasite development through kinetically distinct effects on mosquito innate immune responses. Specifically, knockdown of AsILP4 induced early expression of immune effector genes (1-6 h after infection), a pattern associated with significantly reduced parasite abundance prior to invasion of the midgut epithelium. In contrast, knockdown of AsILP3 increased later expression of the same genes (24 h after infection), a pattern that was associated with significantly reduced oocyst development. These data suggest that P. falciparum parasites alter the expression of mosquito AsILPs to dampen the immune response and facilitate their development in the mosquito vector.

CD8+ T cell-derived IFN-γ prevents infection by a second heterologous virus.

Persistent viral infection is often associated with dysfunctional immune responses against unrelated pathogens. Lymphocytic choriomeningitis virus (LCMV) can establish acute or chronic infections in mice and is widely used as a model for persistent virus infections in humans. Mice infected with LCMV develop a transient defect in Ag-specific immunity against heterologous viral infection. Although it has been proposed that LCMV infection induces an immunosuppressed state within the host, our data show that infected mice successfully clear vaccinia virus through a mechanism that involves CD8(+) T cell-derived IFN-γ. This observation demonstrates that chronic LCMV infection does not impair protective immunity against heterologous viral challenge. Rather, a natural sterilizing immunity is induced following a primary infection that prevents a secondary infection. Our findings suggest a need to re-evaluate current thoughts about the immune suppression that might occur during a persistent infection.