Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

<p>An estimated twenty-five percent of the fifty-seven million annual deaths worldwide can be directly attributed to infectious disease. Mammals contain both adaptive and innate immune systems to deal with invading pathogens. The genetic model organism <em>Caenorhabditis elegans</em> lacks an adaptive immune system, which makes it a powerful model organism to study the innate immune system without the added complexity of an adaptive immune system. Multiple human pathogens can cause lethal infections in <em>C. elegans</em> and several <em>C. elegans</em> innate immune pathways have been identified that are conserved with mammals and protect the nematode from infection. The goal of this work was to identify novel bacterial virulence factors and innate immune defenses against pathogens by using the genetic model organism <em>C. elegans</em>. We established <em>C. elegans</em> as a model for <em>Yersinia pestis</em> infection and used this model to identify novel bacterial virulence factors that were also important for virulence in a mammalian model of infection. Previous studies demonstrated that <em>C. elegans</em> can identify bacterial pathogens using sensory neurons and activate an avoidance response that requires components of G-protein signaling pathways. We screened forty <em>C. elegans</em> strains containing mutations in chemosensory G-protein coupled receptors for altered survival on pathogen and identified <em>npr-1</em> to be required for full <em>C. elegans</em> defense against pathogens. We found that activation of the NPR-1 nervous circuit enhances host susceptibility to microbial infection while inhibition of the circuit boosts innate immunity. This data provides the first evidence that innate immunity in <em>C. elegans</em> is directly linked to the nervous system and establishes the nematode as a novel system to study neuroimmunology. From this work, we have identified <em>Y. pestis</em> virulence-related genes and <em>C. elegans</em> innate immune effector genes required for innate immunity to human bacterial pathogens.</p> / Dissertation

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation. In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at low levels in resting NK cells and three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the production of IFNγ, TNFα and TNFβ through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFNγ and TNFα but does not alter cytotoxicity, whereas over-expression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. To further delineate the targets of PRDM1-mediated regulation in NK cells, global approaches were utilized. Experiments utilizing chromatin immunoprecipitation coupled to promoter tiling arrays identified 292 novel direct targets of PRDM1 binding. These studies revealed widespread binding of PRDM1 to the genome, which was not limited to proximal promoter regions. Furthermore, microarray analysis of stimulated NK cells combined with PRDM1 knockdown has enabled the identification of genes responsive to PRDM1 knockdown using primary cells. Collectively, these experiments identify both direct and indirect targets of PRDM1 regulation and help define a PRDM1-centered gene regulatory network in NK cells. Data presented in the final chapter pertains to an independent project aimed at identifying small molecule inhibitors of the methyltransferase G9a, which is recruited by PRDM1 and is required for silencing of target genes. A mass spectrometry-based assay was developed and used to screen a small molecule library. Several hits were identified and combinatorial chemistry yielded several compounds with < 20µM IC50 values. In cell-based assays, however, treatment with the small molecules had limited efficacy, indicating additional chemical modifications are necessary to yield bioactive compounds. The data presented here demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.

Respiratory syncytial virus (RSV) infection causes ~64 million cases of respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in patients of all ages but strikes the age extremes, infants and the elderly, with highest frequency presumably due to innate immune deficiencies. A higher morbidity and mortality has been reported for the elderly above 65 years of age, which has been attributed to immune senescence. Efforts to generate an effective vaccine have thus far been unsuccessful. The innate immune system provides the first line of defense against viral pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise mechanism of subversion of innate immunity in young and aged is poorly understood. A better understanding of innate immune pathways is expected to aid in the development of appropriate vaccines or prophylactics for these high-risk groups. Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN responses by disrupting components of the innate immune system, although the mechanism is not well defined. We hypothesized that NS1 targets constituents of the PRR pathways to evade innate immunity and thus ensure viral survival. Using microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was also correlated with upstream IFN-response regulator, LGP2, and its expression was inducible in the absence of a viral infection. Tetracycline-inducible expression of recombinant NS1 in a cell model also promoted viral replication and emphasizes the key contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism for RSV NS1 in the disruption of early innate responses through mitochondrial localization and alteration of the RLH signaling. Whereas the above studies showed the importance RSV-induced innate immune pathways, whether the expression and signaling of innate immune pathways were adversely affected upon RSV infection in the high-risk groups remains unknown. Since elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced pneumonia, often resulting in hospitalization and medical intervention and adaptive immune cell functionality and responsiveness reportedly decline with age, we hypothesized a similar age-related deterioration of the innate antiviral system. In this investigation, we used an aged mouse model to correlate age-associated changes in innate immune gene expression with RSV pathology. Of 84 antiviral genes examined, five genes including RIG-I, IFNAR1, TLR8, IL-1Β, and osteopontin (OPN) were associated with both age and infection. In response to RSV infection, aged mice had delayed induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8 agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had increased cellular infiltration and prolonged infection as compared to young mice. In summary, age-related decline in expression and functionality of antiviral defenses were correlated with enhance RSV-induced lung disease in aged mice. In the absence of infection, aged mice chronically overproduced IL-1Β and OPN relative to young mice. Upon infection, aged mice had impaired ability to secrete higher levels of IL-1Β and mucus. In contrast, OPN secretion remained high and prolonged in aged mice throughout infection. The age-related decline in host antiviral gene induction and delayed cytokine production correlated with enhanced disease pathology. Using a transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs. Therefore, OPN overproduction and defective mucus production likely contribute to pathology in aged mice. These findings demonstrate that RSV targets the innate virus recognition and antiviral cytokine activation pathways but also that the antiviral defense system is significantly affected by age. Consequently, efforts to generate vaccines or develop therapies that stimulate IFN induction may prove unsuccessful in the elderly given that RSV virulence factors and age weaken these responses. This study contributes to our understanding of how aging relates to the RSV subversion of the host antiviral response and should help with the development of better antiviral therapies suited to the growing elderly population.

When a patient is exposed surgical stress, the endocrine system secretes hormones in response to that stress. These hormones further activate the immune system to release cytokines and other acute phase reactions. These processes are supposed to protect the body by upregulating the innate immune system and producing an inflammatory response that acts to protect and heal. However, uncontrolled surgical stress may cause a weaker immune response that may lead to delayed wound healing. The phenomenon of unplanned perioperative hypothermia is known to expose patients to additional surgical stress. The purpose of this preliminary experimental study was to determine the effect of a preoperative warming intervention on the acute phase response of surgical stress in surgical patients. Specifically, the aim of this study was to evaluate the effect of a prewarming intervention using a forced-air warming (FAW) device versus routine care (RC) using warmed cotton blankets on the development of unplanned hypothermia, cytokine production, and endocrine responses. It was hypothesized that 1) the FAW participants would experience less unplanned perioperative hypothermia than the RC participants; 2) the FAW participants would experience lower catecholamine and cortisol levels than the RC participants; and 3) the FAW participants would experience higher proinflammatory cytokine and CRP production intra- and postoperatively than the RC participants. Infrared tympanic temperatures and 4 blood samples were taken at 4 time intervals from each of the 28 (n = 14 each group) randomized participants that underwent routine general anesthesia surgery. Serum concentrations of CRP, cortisol and IL-1beta, IL- 6, TNF-alpha, and IFN-gamma, and plasma concentrations of epinephrine and norepinephrine were measured. To test the hypotheses across time and between groups, a repeated measures ANOVA design was used. Though FAW was not associated with a differential endocrine or inflammatory response in this small, preliminary study, further study of forced air warming as a preoperative nursing intervention is warranted. The finding of higher than expected IL-6 levels in the preoperative period suggests a potential role for anxiety, an important factor in psychoneuroimmunological pathways, that could affect recovery and healing. The relationship between surgical stress, anxiety, and preoperative IL-6 deserves further study.

2'-5'-oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding to viral double-stranded RNAs, OAS enzymes produce 2'-5'-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to West Nile virus (WNV) infection, highlighting the importance of the OAS1 enzyme. Here I report that the 5'-terminal region (5'-TR) of the WNV genome, comprising both the 5'-untranslated region (5'-UTR) and initial coding region, is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I (SLI) is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. Solution conformations of both the 5'-TR RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. I also report that the 3' terminal region (3'-TR) is able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-TR that is sufficient for activation of the enzyme. The solution confirmation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-TR in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-TRs, a required step for replication, is not sufficient to protect WNV from OAS1 recognition. The purity, monodispersity and homogeneity of all samples subjected to SAXS analysis were evaluated using dynamic light scattering and/or analytical ultra-centrifuge. These data provide a framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme. / October 2015

Cells detect and respond to environmental changes using intracellular networks, and defects in the wiring of these networks contribute to diseases. For example, Toll-like receptors (TLRs) sense microbial molecules and trigger pathways critical for host defense. Genetic defects in components of the TLR and other pathogen-sensing pathways have been linked to human diseases. Hence, rational targeting of these pathways should help to manipulate immune responses associated with infections, autoimmunity, or vaccines. A fundamental challenge is to dissect the intracellular networks mobilized by pathogen-sensing pathways. Here we present approaches to dissect the TLR network of innate immune dendritic cells (DCs), focusing on two regulatory layers: signaling and transcription. First, we present a strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We apply this approach to derive regulatory networks that control the transcriptional response to TLR engagement by microbial molecules. Our approach revealed the regulatory functions of 125 transcription factors (TFs), chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 “fine-tuners” that help explain how TLR pathways achieve specificity. Second, we report the systematic discovery of signaling components in TLR responses. By combining transcriptional profiling, genetic and small molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known members of the TLR signaling pathways. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Lastly, we expand these approaches to integrate functional and physical interactions linking the ‘signaling-to-transcription’ TLR network. By combining our perturbation-based approach with measurements of physical interactions, including phosphorylation, protein complexes, and TF binding to DNA, we uncover 30 signaling regulators mechanistically linked to 19 downstream TFs. The integration of these datasets into a model reveals the organization of the TLR response. Overall, these studies illustrate the power of combining systematic measurements and perturbations to elucidate complex intracellular circuits and discover potential therapeutic targets.

The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.

In multicellular organisms, communication between cells relies on transmitting information across membrane barriers. Different cell types interrogate particular aspects of their surrounding environment through protein receptors that span membranes and upon ligand binding, trigger enzymatic signaling cascades that culminate in the activation of one or more transcription factors. Information transmission is bidirectional, as individual cells must be able to sense unique aspects of their surroundings, relay their specialized knowledge with others, and receive the collective knowledge of surrounding cells and tissues. This two-way communication is particularly important in the innate immune system, where potentially infectious organisms must be readily detected and identified, and their presence communicated to other cells in the vicinity. Because of the rapid generation time of microorganisms, delays between any of these steps - detection, information processing or information transmission - can make the difference between successful control of infection and pathogen outgrowth. For this reason, the receptors that identify potential pathogens must be able to detect pathogens wherever they are found, be exquisitely sensitive, and initiate a robust response. At the same time, the inflammatory response to infection is itself damaging. This requires that the same receptors are tightly controlled, both by modulating their sensitivity and by rapidly turning off responses through negative feedback pathways. Here, I show that the toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) plays a critical role in controlling the sensitivity of toll-like receptor (TLR) signaling. First, TIRAP controls the assembly of the myddosome, a protein complex that activates signal transduction, from both the plasma membrane and within endosomes of macrophages. Though TIRAP's role at the cell surface was previously described, its endosomal function was previously unknown. Second, TIRAP is an important target for negative regulation. After stimulation with the TLR4 ligand lipopolysaccharide (LPS), macrophages induce a state known as endotoxin tolerance, in which they are refractory for additional LPS stimulation. Many mechanisms for endotoxin tolerance have been proposed, but here I show that TIRAP is degraded in endotoxin tolerance, and that the mechanism of TIRAP degradation also has implications for viral/bacterial superinfection.

Chlamydia trachomatis (CT) is a pestilent infection affecting upwards of 90 million people worldwide. An efficacious vaccine is needed to control the morbidities and rising healthcare cost associated with genital CT infection. We have established that protection against chlamydia infection parallels with a high frequency of T helper Type 1 cells and the associated antibodies. The current study focuses on the induction of innate immune responses involved during Chlamydia infection by a Vibrio cholera ghost-based (VCG) vaccine vector. THP-1 cells were used for dose and kinetic experiments. HeLa cells were used for infectivity assays. Based on preliminary studies, we hypothesized that the induction of immune responses by a VCG-based vaccine involves multiple innate immune signaling. Multiplex assay was used to measure T helper Type I and Type II cytokine secretion by THP-1 monocytes (Mn) or macrophages (Mϕ). Immunostimulatory cytokine secretion was significant when both cell morphologies were pulsed with VCG or VCG/murine splenocytes. We concluded that this secretion was significant enough to compliment that which would be secreted when THP-1 cells are pulsed with Chlamydia elementary bodies alone, enhancing the innate immune response during infection. Cellular supernatants (conditioned media) containing Th1-type and Th2-type cytokines were used to culture Chlamydia-infected HeLa cell monolayers. Infected HeLa monolayers cultured in the conditioned media were significantly less infected (968 IFUs) versus HeLa monolayers cultured in Earle’s minimum essential media (16,486 IFUs; p<0.001). We concluded that factors contained in conditioned media prevent and/or significantly reduce infection by Chlamydia and the development of inclusion forming units.