STING dependent BAX-IRF3 signaling results in apoptosis during late-stage Coxiella burnetii infection.

STING (STimulator of Interferon Genes) is a cytosolic sensor for cyclic dinucleotides (CDNs) and initiates an innate immune response upon binding to CDNs. Coxiella burnetii is a Gram-negative obligate intracellular bacterium and the causative agent of the zoonotic disease Q fever. The ability of C. burnetii to inhibit host cell death is a critical factor in disease development. Previous studies have shown that C. burnetii inhibits host cell apoptosis at early stages of infection. However, during the late-stages of infection, there is host cell lysis resulting in the release of bacteria to infect bystander cells. Thus, we investigated the role of STING during late-stages of C. burnetii infection and examined STING's impact on host cell death. We show that the loss of STING results in higher bacterial loads and abrogates IFNß and IL6 induction at 12 days post-infection. The absence of STING during C. burnetii infection significantly reduces apoptosis through decreased caspase-8 and -3 activation. During infection, STING activates IRF3 which interacts with BAX. BAX then translocates to the mitochondria, which is followed by mitochondrial membrane depolarization. This results in increased cytosolic mtDNA in a STING-dependent manner. The presence of increased cytosolic mtDNA results in greater cytosolic 2'-3' cGAMP, creating a positive feedback loop and leading to further increases in STING activation and its downstream signaling. Taken together, we show that STING signaling is critical for BAX-IRF3-mediated mitochondria-induced apoptosis during late-stage C. burnetii infection.

Drosophila melanogaster Sting mediates Coxiella burnetii infection by reducing accumulation of reactive oxygen species.

The Gram-negative bacterium Coxiella burnetii is the causative agent of query fever in humans and coxiellosis in livestock. C. burnetii infects a variety of cell types, tissues, and animal species including mammals and arthropods, but there is much left to be understood about the molecular mechanisms at play during infection in distinct species. Human stimulator of interferon genes (STING) induces an innate immune response through the induction of type I interferons (IFNs), and IFN promotes or suppresses C. burnetii replication, depending on tissue type. Drosophila melanogaster contains a functional STING ortholog (Sting) which activates NF-κB signaling and autophagy. Here, we sought to address the role of D. melanogaster Sting during C. burnetii infection to uncover how Sting regulates C. burnetii infection in flies. We show that Sting-null flies exhibit higher mortality and reduced induction of antimicrobial peptides following C. burnetii infection compared to control flies. Additionally, Sting-null flies induce lower levels of oxidative stress genes during infection, but the provision of N-acetyl-cysteine (NAC) in food rescues Sting-null host survival. Lastly, we find that reactive oxygen species levels during C. burnetii infection are higher in Drosophila S2 cells knocked down for Sting compared to control cells. Our results show that at the host level, NAC provides protection against C. burnetii infection in the absence of Sting, thus establishing a role for Sting in protection against oxidative stress during C. burnetii infection.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria which causes the global zoonotic disease Q Fever. Treatment options for infection are limited, and development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected TNFα/CHX-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- BMDMs to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

An impaired ubiquitin-proteasome system increases APOBEC3A abundance.

Apolipoprotein B messenger RNA (mRNA) editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases cause genetic instability during cancer development. Elevated APOBEC3A (A3A) levels result in APOBEC signature mutations; however, mechanisms regulating A3A abundance in breast cancer are unknown. Here, we show that dysregulating the ubiquitin-proteasome system with proteasome inhibitors, including Food and Drug Administration-approved anticancer drugs, increased A3A abundance in breast cancer and multiple myeloma cell lines. Unexpectedly, elevated A3A occurs via an ∼100-fold increase in A3A mRNA levels, indicating that proteasome inhibition triggers a transcriptional response as opposed to or in addition to blocking A3A degradation. This transcriptional regulation is mediated in part through FBXO22, a protein that functions in SKP1-cullin-F-box ubiquitin ligase complexes and becomes dysregulated during carcinogenesis. Proteasome inhibitors increased cellular cytidine deaminase activity, decreased cellular proliferation and increased genomic DNA damage in an A3A-dependent manner. Our findings suggest that proteasome dysfunction, either acquired during cancer development or induced therapeutically, could increase A3A-induced genetic heterogeneity and thereby influence therapeutic responses in patients.

Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species, we sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. Here, we identify insulin-mediated endothelin signaling using the D. melanogaster model and evaluate an orthologous pathway in human cells during WNV infection. We demonstrate that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, our findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease. IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identifies potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

The Unfolded-Protein Response Triggers the Arthropod Immune Deficiency Pathway.

The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.

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.

To die or not to die: Programmed cell death responses and their interactions with Coxiella burnetii infection.

Coxiella burnetii is a Gram-negative, obligate intracellular, macrophage-tropic bacterium, and the causative agent of the zoonotic disease Q fever. The epidemiology of Q fever is associated with the presence of infected animals; sheep, goats, cattle, and humans primarily become infected by inhalation of contaminated aerosols. In humans, the acute phase of the disease is characterized primarily by influenza-like symptoms, and approximately 3%-5% of the infected individuals develop chronic infection. C. burnetii infection activates many types of immune responses, and the bacteria's genome encodes for numerous effector proteins that interact with host immune signaling mechanisms. Here, we will discuss two forms of programmed cell death, apoptosis, and pyroptosis. Apoptosis is a form of non-inflammatory cell death that leads to phagocytosis of small membrane-bound bodies. Conversely, pyroptosis results in lytic cell death accompanied by the release of proinflammatory cytokines. Both apoptosis and pyroptosis have been implicated in the clearance of intracellular bacterial pathogens, including C. burnetii. Finally, we will discuss the role of autophagy, the degradation of unwanted cellular components, during C. burnetii infection. Together, the review of these forms of programmed cell death will open new research questions aimed at combating this highly infectious pathogen for which treatment options are limited.

Metabolic Plasticity Aids Amphotropism of Coxiella burnetii.

Coxiella burnetii, the causative agent of query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms (e.g., mammals and arthropods) and cell types. This amphotropic nature of C. burnetii, in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, CbΔpckA, unable to undergo gluconeogenesis. Both the parental strain and CbΔpckA mutant exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, interstitial fluid-modeled medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by the CbΔpckA mutant in specific cell lines. The CbΔpckA mutant was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen.

Host Factors That Control Mosquito-Borne Viral Infections in Humans and Their Vector.

Mosquito-borne viral infections are responsible for a significant degree of morbidity and mortality across the globe due to the severe diseases these infections cause, and they continue to increase each year. These viruses are dependent on the mosquito vector as the primary means of transmission to new vertebrate hosts including avian, livestock, and human populations. Due to the dynamic host environments that mosquito-borne viruses pass through as they are transmitted between vector and vertebrate hosts, there are various host factors that control the response to infection over the course of the pathogen's life cycle. In this review, we discuss these host factors that are present in either vector or vertebrate models during infection, how they vary or are conserved between hosts, and their implications in future research pertaining to disease prevention and treatment.

Natural genetic variation in Drosophila melanogaster reveals genes associated with Coxiella burnetii infection.

The gram-negative bacterium Coxiella burnetii is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Host genetics are associated with C. burnetii pathogenesis both in humans and animals; however, it remains unknown if specific genes are associated with severity of infection. We employed the Drosophila Genetics Reference Panel to perform a genome-wide association study to identify host genetic variants that affect host survival to C. burnetii infection. The genome-wide association study identified 64 unique variants (P < 10-5) associated with 25 candidate genes. We examined the role each candidate gene contributes to host survival during C. burnetii infection using flies carrying a null mutation or RNAi knockdown of each candidate. We validated 15 of the 25 candidate genes using at least one method. This is the first report establishing involvement of many of these genes or their homologs with C. burnetii susceptibility in any system. Among the validated genes, FER and tara play roles in the JAK/STAT, JNK, and decapentaplegic/TGF-ß signaling pathways which are components of known innate immune responses to C. burnetii infection. CG42673 and DIP-ε play roles in bacterial infection and synaptic signaling but have no previous association with C. burnetii pathogenesis. Furthermore, since the mammalian ortholog of CG13404 (PLGRKT) is an important regulator of macrophage function, CG13404 could play a role in host susceptibility to C. burnetii through hemocyte regulation. These insights provide a foundation for further investigation regarding the genetics of C. burnetii susceptibility across a wide variety of hosts.

Antinociceptive and Immune Effects of Delta-9-Tetrahydrocannabinol or Cannabidiol in Male Versus Female Rats with Persistent Inflammatory Pain.

Chronic pain is the most common reason reported for using medical cannabis. The goal of this research was to determine whether the two primary phytocannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are effective treatments for persistent inflammatory pain. In experiment 1, inflammation was induced by intraplantar injection of Complete Freund's adjuvant (CFA). Then THC (0.0-4.0 mg/kg, i.p.) or CBD (0.0-10 mg/kg, i.p.) was administered twice daily for 3 days. On day 4, THC, CBD, or vehicle was administered, and allodynia, hyperalgesia, weight-bearing, locomotor activity, and hindpaw edema were assessed 0.5-4 hours postinjection. In experiment 2, CFA or mineral oil (no-pain control)-treated rats were given THC (2.0 mg/kg, i.p.), CBD (10 mg/kg, i.p.), or vehicle in the same manner as in experiment 1. Four hours postinjection on day 4, serum samples were taken for analysis of cytokines known to influence inflammatory pain: interleukin (IL)-1ß, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α THC dose-dependently reduced pain-related behaviors but did not reduce hindpaw edema, and little tolerance developed to THC's effects. In contrast, CBD effects on inflammatory pain were minimal. THC produced little to no change in serum cytokines, whereas CBD decreased IL-1ß, IL-10, and IFN-γ and increased IL-6. Few sex differences in antinociception or immune modulation were observed with either drug, but CFA-induced immune activation was significantly greater in males than females. These results suggest that THC may be more beneficial than CBD for reducing inflammatory pain in that THC maintains its efficacy with short-term treatment in both sexes and does not induce immune activation. SIGNIFICANCE STATEMENT: The pain-relieving effects of cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) are examined in male and female rats with persistent inflammatory pain to determine whether individual phytocannabinoids could be a viable treatment for men and women with chronic inflammatory pain. Additionally, sex differences in the immune response to an adjuvant and to THC and CBD are characterized to provide preliminary insight into immune-related effects of cannabinoid-based therapy for pain.

Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors.

The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. Here, we use a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. We determine that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we show that insulin signaling activates the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, we validate that insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens.

Natural Genetic Variation Screen in Drosophila Identifies Wnt Signaling, Mitochondrial Metabolism, and Redox Homeostasis Genes as Modifiers of Apoptosis.

Apoptosis is the primary cause of degeneration in a number of neuronal, muscular, and metabolic disorders. These diseases are subject to a great deal of phenotypic heterogeneity in patient populations, primarily due to differences in genetic variation between individuals. This creates a barrier to effective diagnosis and treatment. Understanding how genetic variation influences apoptosis could lead to the development of new therapeutics and better personalized treatment approaches. In this study, we examine the impact of the natural genetic variation in the Drosophila Genetic Reference Panel (DGRP) on two models of apoptosis-induced retinal degeneration: overexpression of p53 or reaper (rpr). We identify a number of known apoptotic, neural, and developmental genes as candidate modifiers of degeneration. We also use Gene Set Enrichment Analysis (GSEA) to identify pathways that harbor genetic variation that impact these apoptosis models, including Wnt signaling, mitochondrial metabolism, and redox homeostasis. Finally, we demonstrate that many of these candidates have a functional effect on apoptosis and degeneration. These studies provide a number of avenues for modifying genes and pathways of apoptosis-related disease.

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders.

Innate immunity, the first line of defense against invading pathogens, is an ancient form of host defense found in all animals, from sponges to humans. During infection, innate immune receptors recognize conserved molecular patterns, such as microbial surface molecules, metabolites produces during infection, or nucleic acids of the microbe's genome. When initiated, the innate immune response activates a host defense program that leads to the synthesis proteins capable of pathogen killing. In mammals, the induction of cytokines during the innate immune response leads to the recruitment of professional immune cells to the site of infection, leading to an adaptive immune response. While a fully functional innate immune response is crucial for a proper host response and curbing microbial infection, if the innate immune response is dysfunctional and is activated in the absence of infection, autoinflammation and autoimmune disorders can develop. Therefore, it follows that the innate immune response must be tightly controlled to avoid an autoimmune response from host-derived molecules, yet still unencumbered to respond to infection. In this review, we will focus on the innate immune response activated from cytosolic nucleic acids, derived from the microbe or host itself. We will depict how viruses and bacteria activate these nucleic acid sensing pathways and their mechanisms to inhibit the pathways. We will also describe the autoinflammatory and autoimmune disorders that develop when these pathways are hyperactive. Finally, we will discuss gaps in knowledge with regard to innate immune response failure and identify where further research is needed.

Emerging Mechanisms of Insulin-Mediated Antiviral Immunity in Drosophila melanogaster.

Arboviruses (arthropod-borne viruses), such as Zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, include some of the most significant global health risks to human populations. The steady increase in the number of cases is of great concern due to the debilitating diseases associated with each viral infection. Because these viruses all depend on the mosquito as a vector for disease transmission, current research has focused on identifying immune mechanisms used by insects to effectively harbor these viruses and cause disease in humans and other animals. Drosophila melanogaster are a vital model to study arboviral infections and host responses as they are a genetically malleable model organism for experimentation that can complement analysis in the virus' natural vectors. D. melanogaster encode a number of distinct mechanisms of antiviral defense that are found in both mosquito and vertebrate animal systems, providing a viable model for study. These pathways include canonical antiviral modules such as RNA interference (RNAi), JAK/STAT signaling, and the induction of STING-mediated immune responses like autophagy. Insulin signaling plays a significant role in host-pathogen interactions. The exact mechanisms of insulin-mediated immune responses vary with each virus type, but nevertheless ultimately demonstrates that metabolic and immune signaling are coupled for antiviral immunity in an arthropod model. This mini review provides our current understanding of antiviral mechanisms in D. melanogaster, with a focus on insulin-mediated antiviral signaling, and how such immune responses pertain to disease models in vertebrate and mosquito species.

Astrocyte expression of the Drosophila TNF-alpha homologue, Eiger, regulates sleep in flies.

Sleep contributes to cognitive functioning and is sufficient to alter brain morphology and function. However, mechanisms underlying sleep regulation remain poorly understood. In mammals, tumor necrosis factor-alpha (TNFα) is known to regulate sleep, and cytokine expression may represent an evolutionarily ancient mechanism in sleep regulation. Here we show that the Drosophila TNFα homologue, Eiger, mediates sleep in flies. We show that knockdown of Eiger in astrocytes, but not in neurons, significantly reduces sleep duration, and total loss-of-function reduces the homeostatic response to sleep loss. In addition, we show that neuronal, but not astrocyte, expression of the TNFα receptor superfamily member, Wengen, is necessary for sleep deprivation-induced homeostatic response and for mediating increases in sleep in response to human TNFα. These data identify a novel astrocyte-to-neuron signaling mechanism in the regulation of sleep homeostasis and show that the Drosophila cytokine, Eiger, represents an evolutionarily conserved mechanism of sleep regulation across phylogeny.