Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process. We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis. Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important. Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.

Innate immunity relies on a repertoire of germline-encoded non-rearranging pattern recognition receptors that bind to invariant microbial surface molecules. This event initiates a number of signal transduction cascades that lead to humoral and cellular defense responses like synthesis of antimicrobial peptides, phagocytosis and coagulation – mechanisms that efficiently fight infectious microorganisms and have been evolutionary conserved to exist in parallel with the antibody-based adaptive immunity found in vertebrates. The fruit fly, Drosophila melanogaster represents a widely used animal model for studies of a pristine innate immune system. Its immune responsive intracellular signalling pathways display a high degree of similarity with the NF-κB /Rel-signalling pathways that mediate the inflammatory response in mammals. Insects are also vectors for medically important parasitic diseases which can trigger immune responses in the vector so basal knowledge about the regulation and function of insect immune systems can contribute to our understanding of inflammation and microbial disease in higher animals and open new strategies for biological vector control. Drosophila hemocytes play a key role in executing and coordinating local and systemic defenses in response to infection. This thesis describes in vitro studies of Drosophila gene expression in response to bacterial infection using the larval hemocyte-like cell line – mbn-2. Our results show that immune challenge with bacterial cell wall components and intact live bacteria induces differential gene expression that gives clues to how cellular immune responses could be activated and regulated.

Viruses are very abundant in water and hence diseases caused by viruses are common in marine organisms. These diseases create great problems for the commercial farming of crustaceans and mussels. One of the most common and most disastrous diseases for shrimp is caused by the white spot syndrome virus (WSSV), which is spread all around the world and also is infecting many different species of crustaceans including freshwater crayfish. Although during recent years knowledge has been gathered on the ways in which invertebrates defend themselves against bacteria and fungi virtually nothing is known about the defence processes elicited by virus. The aim of this work was to develop a model to use for studies of virus-host interactions in vivo and in vitro. Temperature was found to be important for the virus infectivity and at lower temperature the virus apparently did not replicate, but if animals kept at low temperature for more than 40 days were transferred to higher temperatures they died quickly due to an increased virus replication. In crayfish infected with the virus it was found that hemocytes did not degranulate and the melanization reaction was also inhibited in the hemocyes. Thus it is apparent that this virus interacts with the immune system and hemocytes in particular and to be able to study this in some greater detail it was necessary to develop a cell culture to study virus-host interactions at the molecular level. Hence, we have developed a stem cell culture from the hematopoietic tissue (hpt) that will differentiate and mature into hemocytes and which can be used to replicate the WSSV in the presence of an endogenous cytokine, astakine. Astakine is the first cytokine like-factor described which is directly involved in hematopoiesis in an invertebrate.

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

All eukaryotic organisms have an innate immune system characterized by germ-line encoded receptors and effector molecules, which mediate detection and clearance of microbes such as bacteria, fungi, and parasites. VertebrateDrosophila as a genetically tractable organism with a This thesis concerns the peptidoglycan recognition protein (PGRP) gene family in the fruit fly. The family consists of thirteen genes, of which a few have been reported to be part of the signaling pathways that regulates immune Data presented show that the putative receptors have affinity for peptidoglycan, but not for lipopolysaccharide, or the fungal cell wall polymer beta-glucan. PGRP-SA, receptor of the Toll pathway, has a preference for In a search for novel PGRP receptors I found two PGRP proteins that instead displayed enzymatic activity towards peptidoglycan. They are of the N-actylmuramoyl L-alanine amidase type, which degrades peptidoglycan by splittingStaphylococcus aureus peptidoglycan looses its immune elicitor capacity. This is in contrast to lysozyme-degraded peptidoglycan, which isDrosophila PGRPs to be potential enzymes. PGRP-SB1 is the other enzymatic PGRP described within this thesis. It has a moreBacillus megaterium. In conclusion, receptor PGRP proteins binds bacterial peptidoglycan and triggers immune gene pathways and enzymatic PGRPs have the capacity to reduce the elicitor property of peptidoglycan.

In general the work behind this thesis has revolved around the interesting pattern recognition gene family PGRPs (peptidoglycan recognition proteins). In particular the transmembrane PGRP-LC and to investigate its multifaceted role in the immune response of the fruit fly. As a well characterized model organism living on, and surrounded by, a multitude of microorganisms, Drosophila melanogaster serves as a great tool to gain insights about innate immunity. The two pillars of Drosophila innate immunity are the humoral and the cellular defense. Together they are very potent and can vanquish many infections, but if one of these pillars is damaged, chances are that the defense will collapse and the organism will succumb to the infection. The initial step in any immune response is to become aware of the pathogen. To accomplish this, innate immunity relies on recognizing common molecular building blocks necessary each group of microorganisms. One such building block is the bacterial cell wall component peptidoglycan. PGRPs are a widely spread gene family, and proteins of this family can bind peptidoglycan. We describe that there are 13 PGRP genes in Drosophila, one these codes for PGRP-LC. As it sits in the cell membrane in any of its three different splice forms, PGRP-LC can bind peptidoglycan, dimerize, and subsequently activate the imd/relish signalling pathway, and thereby trigger a vast production of antimicrobial peptides. These short peptides are the firearms of the humoral response. We identified three new inducible antimicrobial peptide genes, Diptericin B, Attacin C and Attacin D. Analyses of their sequences shed light on the evolution and relationship of these antimicrobial peptides The antimicrobial peptides are potent weapons, but without a functional cellular response the animal is at loss. Animals lacking blood cells are gravely compromised. It is interesting to find that PGRP-LC is involved at this end of the immune response equation as well. We have found that PGRP-LC is able to activate blood cells and increase numbers of circulating cells, in a JNK (Jun N-terminal kinase) dependent manner. Intriguingly this activation is not dependent on Relish, the NF-kB transcription factor of the Imd/Relish pathway. PGRP-LC activation funnels into both Imd/Relish and the JNK pathways. When PGRP-LC is lost, it appears that some basal, or background, JNK activation is lost. These effects are very mild, however the animal appears to become more sensitive to additional perturbations in this signalling pathway. This was the starting point when we started to re-evaluate Dredd, the caspase responsible for cleaving and activating Relish. Dredd also contributes to the JNK signalling pathway.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

<p>Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.</p><p>IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (<italic>Il1r1</italic>-/-) mice given <italic>Il1r1</italic>-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of <italic>Il1r1</italic>-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given <italic>Il1r1</italic>-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.</p> / Dissertation

Cardiovascular disease, and myocardial infarction (MI) in particular, remains a major burden in the developed world today. In fact, the remodeling process, which follows the initial ischemic episode of MI, is a major determinant of heart failure. Although several key mechanistic pathways involving cell growth and death have been identified, there is limited knowledge surrounding the role of the innate immune response as a positive or negative regulator of cardiac remodeling. Recent data strongly support a role for key regulatory components within the toll-like receptor (TLR) family as potent modulators of cardiac remodeling post-MI. It has been demonstrated that targeted gene knockdown of TLR4, as well as downstream adaptor proteins and kinases, significantly improve cardiac function and overall survival. While the well-known NF-κB transcriptional factor that is downstream to TLR4 signaling has been linked to remodeling, there has been no evidence thus far describing a role of the parallel interferon regulatory factor-3 (IRF3) signaling cascade in any facet of this process. Several key findings suggest that IRFs contribute to both cell growth and apoptosis, thus providing an appealing, and novel target for interrogation. In this thesis I describe how IRF3 contributes to maladaptive remodeling post-MI. In my first set of experiments, I demonstrate that IRF3 is acutely upregulated within the cardiomyocyte following MI and that this response contributes to excessive apoptosis post-MI. A targeted deletion of the IRF3 gene enhances cardiac function, decreases infarct size, and improves survival following MI. In the second set of experiments I demonstrate that IRF3 attenuates angiogenesis at the ischemic border zone by upregulating the expression of thrombospondins. I have shown that IRF3 deficiency, which liberates endogenous anti-angiogenic signals, promotes angiogenesis following ischemic injury. These data suggest that IRF3 is a potent regulator of cardiac remodeling and may be an effective therapeutic target to ameliorate maladaptive cardiac repair post-MI.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF’s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.