The Role of Morphology Transitions in Tissue-to-Blood Spread of Infestation

The fungal organism Candida albicans is a nearly ubiquitous commensal inhabitant of the human body. However, in susceptible individuals it can establish mucosal infections as well as life-threatening systemic infection. We are investigating a key contributor to C. albicans’ pathogenesis: its ability to switch among multiple growth forms in response to an array of environmental signals. We hypothesize that this ability to undergo morphological transitions mediates its ability to disseminate from localized infections to system-wide bloodstream infection. Using a transparent zebrafish embryo model of infection, we have directly assessed specific contributions of C. albicans’ morphologies in the process of tissue-to-bloodstream dissemination. We have observed that yeast is the primary form of C. albicans within the bloodstream. We have demonstrated that hyphae are not required for dissemination in our model, but they play an important role in promoting yeast cell dissemination. It is our expectation that further elucidation of the roles of morphological transitions will permit the development of more effective therapies to prevent C. albicans-related mortality in susceptible individuals

Bacterial and Host Factors Contributing to Mycobacterium avium subspecies paratuberculosis Persistent Infection

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic granulomatous enteritis that plagues domestic and wild ruminants globally. During the silent stages of Johne’s disease, infected animals intermittently shed bacteria for years prior to clinical diagnosis during advanced disease stages. This strategy allows MAP to persist and spread in the environment and on farms undetected leading to enormous financial loss. There is a need for better understanding to understand many aspects of bacterial pathogenesis in order to successfully control and prevent MAP infection. The research of this dissertation aims to identify a) virulence factors of MAP that contribute to pathogen escape of host killing mechanisms and b) host cellular factors that allow MAP to persist and survive within professional phagocytes. We describe Acanthamoeba castellanii (amoeba) as a MAP infection model of the environmental protozoan host and as a tool for selecting bacterial genetic factors potentially contributing to MAP virulence in host macrophages. Using MAP transposon gene knockout mutants, we demonstrate altered amoeba metabolism and that associated metabolic changes, identified through screening of MAP transposon gene knockout mutants, which mirror survival patterns and intracellular MAP burdens during macrophage infection. Transposon mutant infections of amoeba led to -infected amoebas display differential (either excessive or decreased) changes in metabolic activity versus those of the compared to wild-type (WT) MAP infection. The majority of mutants identified in the amoeba screen were also found to be growth deficient in Raw 264.7 macrophages. Sequencing and bioinformatic analysis of these mutants help to understand and further characterize pathogen-associated factors that the pathogen most likely uses as a strategy used to persist and grow within host macrophages. To further our knowledge of host-pathogen interactions, we used an in vitro cell culture passage model previously developed in our lab. MAP was found to develop a more pro-inflammatory phenotype after being passed through passage in bovine epithelial cells and macrophages. Utilizing global proteomic analysis of macrophages to analyze cellular metabolic and regulatory pathways, we found promotion of an intracellular phenotype and adaptation within phagocytic cells during different stages of infection. In this dissertation, we show that direct infection of phagocytes by plate growth MAP (direct infection) leads to a robust pro-inflammatory protein profile, metabolic and cellular targets of anti-inflammatory pathways are being highly synthesized when while MAP is first passaged through epithelial cells and before the infection of prior to infecting macrophages (direct infection) upregulates metabolic and cellular targets of anti-inflammatory pathways. Ultimately, specific protein ontology groups including extracellular matrix proteins (integrins) and intracellular organelle coordination proteins were identified. An integrin binding assay and the transwell migration assay confirm enrichment of ECM protein production in macrophages that lead to increased binding and transmigration through the endothelium monolayer. Together, two modeling systems used in this dissertation identified specific host and pathogen factors that significantly expanded our understanding of bacterial genetic factors and phagocytic cell signaling pathways that may allow extended MAP survival within a host. These findings provide the framework to develop strategies for elimination, prevention and diagnostics of Johne’s disease

Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIb alpha-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.Siirretty Doriast

Neutrophil and endothelial cell-mediated inflammation in abdominal sepsis

Sepsis is defined as a life-threatening condition caused by a dysregulated host response to infection. Neutrophils are themost abundant innate immune cells of the body and play a key role in septic pathogenesis. During sepsis activatedneutrophils release web-like traps decorated with various cellular proteins known as neutrophil extracellular traps(NET). The primary task of NET and NET-associated proteins are to kill pathogens; however, excessive accumulationof NET is known to cause tissue damage. Endothelial cells are important for regulating vascular permeability andbarrier functions; however, during sepsis endothelial cells get activated and contribute to tissue damage andorgan failure. The four original studies included in this thesis aimed to investigate new mechanisms involved information of NET, lung injury and pulmonary endothelial cell activation in abdominal sepsis. In study I, we havefound that c-Abl kinase regulate NET formation through ROS signaling pathway. Blocking of c-Abl kinase notonly inhibited NET formation but also reduced inflammation and tissue damage in sepsis. In study II, weinvestigated the role of actin-related protein 2/3 complex (Arp2/3 complex) and found that it regulates neutrophiltrap expulsion both in vivo and in vitro. Inhibition of Arp2/3 complex not only reduced the neutrophil infiltration inbronchoalveolar space, but also alleviated lung damage in abdominal sepsis. In study III, we investigated the roleof S100A9, a pro-inflammatory alarmin, in regulating inflammation and tissue damage in abdominal sepsis.Inhibition of S100A9 by a specific inhibitor, ABR-238901, decreased sepsis-induced neutrophil activation,cytokine formation as well as damage to the lung tissue. In study IV, we examined global transcriptomic changesin a subgroup of lung endothelial cells during sepsis. We found that sepsis caused transcriptomic changes ofgenes related to regulation of coagulation, vascular permeability as well as wound healing and lipid metabolic incapillary endothelial cells. In contrast, postcapillary venules were found to be more enriched with genes related tochemotaxis, cell-cell adhesion of integrins, chemokine biosynthesis, regulation of actin polymerization andneutrophil homeostasis after sepsis. Together, these results demonstrated that targeting c-Abl, Arp2/3 complex,S100A9 or endothelial functions could be useful targets to ameliorate neutrophil mediated tissue injury in sepsis

Inflammation in the CNS: advancing the field using intravital imaging

Inflammation of the CNS can have devastating, long-lived, and in some cases fatal consequences for patients. The stimuli that can induce CNS inflammation are diverse, and include infectious agents, autoimmune responses against CNS-expressed antigens, and sterile inflammation following ischemia or traumatic injury. In these conditions, cells of the immune system play central roles in promulgation and resolution of the inflammatory response. However, the immunological mechanisms at work in these diverse responses differ according to the nature of the response. Our understanding of the actions of immune cells in the CNS has been restricted by the difficulty in visualising leukocytes as they undergo recruitment from the cerebral microvasculature and following their entry into the CNS parenchyma. However, advances in in vivo microscopy over the last 10-15 years have overcome many of these difficulties, and studies using these forms of microscopy have revealed a wealth of new information regarding the cellular and molecular mechanisms of CNS inflammation. This Research Topic brings together state of the art reviews examining the use of in vivo imaging in investigating inflammation and leukocyte behaviour in the CNS. Papers in this Research Topic describe how in vivo microscopy has increased our understanding of the actions of immune cells in the inflamed CNS, following various stimuli including autoimmunity, infection and sterile inflammation

Atherogenesis

This monograph will bring out the state-of-the-art advances in the dynamics of cholesterol transport and will address several important issues that pertain to oxidative stress and inflammation. The book is divided into three major sections. The book will offer insights into the roles of specific cytokines, inflammation, and oxidative stress in atherosclerosis and is intended for new researchers who are curious about atherosclerosis as well as for established senior researchers and clinicians who would be interested in novel findings that may link various aspects of the disease

Role of translocator protein (18 kDa) (TSPO) in retinal phagocytes in a mouse model of age-related macular degeneration (AMD)

Aberrant immune responses including reactive phagocytes are implicated in the etiology of age-related macular degeneration (AMD), a major cause of blindness in the elderly. Microglia, the resident phagocytes of the retina, play an active role in driving disease onset and progression and thus represent a broad target for therapy. Pharmacological approaches of microglia-related immunomodulation aim at dampening the harmful microglia response while preserving their homeostatic functions. The translocator protein (18kDa) (TSPO) is described as a biomarker for reactive gliosis and specific TSPO ligands have been shown to potently modulate microglia-related inflammatory responses and improved disease outcome in various preclinical model systems including Alzheimer’s, Parkinson’s, multiple sclerosis and degenerative diseases of the retina. However, the underlying molecular mechanisms of TSPO-mediated immunomodulation and its biological functions in health and disease remain elusive. In this study, we report that tamoxifen-induced conditional deletion of TSPO in resident microglia using Cx3cr1CreERT2:TSPOfl/fl mice or targeting the protein with the synthetic ligand XBD173 prevents reactivity of retinal phagocytes in the laser-induced mouse model of neovascular AMD. Concomitantly, the subsequent neoangiogenesis and vascular leakage are also prevented by microglia-specific TSPO knockout or XBD173 treatment. Using different NADPH oxidase (NOX)-deficient mice, we show for the first time that TSPO is a key regulator of NOX1-dependent neurotoxic ROS production in the retina. Here, TSPO regulates the Ca2+ influx from the extracellular milieu into the cytosol that is required for stimulation of NOX1 activity and expression in microglia. We also demonstrate that NOX1-derived ROS induce photoreceptor cell death in a paracrine manner and accordingly, NOX1 knockout mice show the same beneficial effects on CNV and wound healing as XBD173 treatment or microglia-specific TSPO knockout. Taken together, we showed that TSPO acts as a regulatory node and regulates microglia functions through both NOX1-dependent and independent mechanisms, defining a distinct role for TSPO in retinal phagocyte reactivity and highlights the protein as a drug target for immunomodulatory and antioxidant therapies for AMD

Evaluation of 68Ga-labeled PET radiopharmaceuticals for imaging of αvβ3 integrin and vascular adhesion protein-1 in inflammation and cancer

Inflammation is involved in the pathogenesis of several chronic diseases as well as in the development of cancer. Vascular adhesion protein-1 (VAP-1) is an inflammation inducible endothelial adhesion molecule that participates in the leukocyte extravasation from blood to sites of inflammation. Under normal conditions, VAP-1 is absent on the surface of endothelium but, upon inflammation, is rapidly translocated from intracellular storage granules to the endothelial cell surface. Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a leukocyte ligand of VAP-1 and the [68Ga]Ga-DOTA-Siglec-9 is a promising positron emission tomography (PET) radiopharmaceutical for the imaging of inflammation. Another adhesion molecule, αvβ3 integrin, is overexpressed in angiogenic endothelial cells, and is therefore considered a target for the imaging of angiogenesis in tumors and inflammatory diseases. The aim of the studies included in this thesis was 1) to evaluate the feasibility of [68Ga]Ga-DOTA-Siglec-9 for the imaging of inflammation in mouse models of arthritis and melanoma, and 2) to explore its safety, whole-body distribution, and radiation dosimetry in healthy subjects as well as its ability to detect arthritis in a patient with rheumatoid arthritis. In mouse melanoma studies, in addition to VAP-1, αvβ3 integrin activation was studied using [68Ga]Ga-DOTA-E[c(RGDfK)]2. The in vivo PET imaging, ex vivo gamma counting, tissue autoradiography, and histological and immunohistochemical stainings were utilized in these studies. Dynamic PET/computed tomography (CT) imaging with concurrent blood sampling clarified the whole-body distribution kinetics, targeting, and radiation exposure of [68Ga]Ga-DOTA-Siglec-9 in humans. The results showed that [68Ga]Ga-DOTA-Siglec-9 clearly detected inflammation in the mouse arthritis and melanoma models, and longitudinal PET/CT allowed the monitoring of disease development over time. In mouse melanoma, [68Ga]Ga- DOTA-E[c(RGDfK)]2 detected changes of αvβ3 integrin expression and activity. In humans, the [68Ga]Ga-DOTA-Siglec-9 was safe and well-tolerated, and capable of detecting arthritic joints. In conclusion, these preclinical and clinical studies indicate that [68Ga]Ga-DOTA-Siglec-9 is a promising new PET radiopharmaceutical for imaging inflammation. In the future, [68Ga]Ga-DOTA-Siglec-9 PET may have potential for imaging various inflammatory diseases besides rheumatoid arthritis