Necroptosis, a type of regulated necrotic cell death, involves cell membrane permeabilization and has been implicated in various acute and chronic pro-inflammatory diseases, including ischemia-reperfusion injury and neurodegenerative diseases. By using in vitro reconstitution studies and a chemical inhibitor, the kinase activity of the serine/threonine kinase RIPK1 had been shown to regulate necroptotic signaling downstream of TNF and Toll-like receptors (TLRs). To investigate the contribution of RIPK1 kinase activity to inflammation and necroptosis in vivo, we generated kinase inactive RIPK1 knock-in mice. Utilizing fibroblasts and macrophages from these mice, we demonstrate that RIPK1 kinase activity is required for necroptotic complex formation and death induction downstream of TNFR1 and TLRs 3 and 4. We show that RIPK1 kinase inactive mice are resistant to TNF-induced shock and exhibit impaired upregulation of TNF-induced cytokines and chemokines in vitro and in vivo. By using bone marrow reconstitution experiments, we demonstrate that RIPK1 kinase activity in a non-hematopoietic lineage drives TNF-induced lethality. We establish that RIPK1 kinase activity is required for TNF-induced increases in intestinal and vascular permeability and clotting, and implicate endothelial cell necroptosis as an underlying factor contributing to TNF/zVAD-induced shock. Thus, work in this thesis reveals that RIPK1 kinase inhibitors may have promise in treating shock and sepsis.

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(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...

The aim of the present study was to find out whether adaption to chronic hypoxia affects the expresion of TNF-α and IL-10 in rat myocardium. TNF-α is a proinflammatory cytokine, which amplifies inflammatory reaction, while IL-10 has opposite antiinflammatory effect. We also measured concentration of nitrotyrosine as a marker of nitrosative stress. We used male Wistar rats divided into four groups: 1) normoxic controls; 2) exposed to continous normobaric hypoxia (10% O2) for three days or 3) for three weeks and 4) exposed to intermittent normobaric hypoxia (10% O2) for three weeks with one hour daily reoxygenation. Cytosolic and membrane proteins (cytosolic and particulate fractions) were obtained from the left ventricle, right ventricle and interventricular septum. Concentrations of TNF-α and IL-10 in both fractions were measured by ELISA. Continous hypoxia increased TNF-α production in particulate fractions from all ventricular parts and decreased the ratio of IL-10/TNF-α in particulate and cytosolic fractions. Intermittent hypoxia redistributed TNF-α from cytosol into the particulate fraction and prevented the drop of IL-10/TNF-α ratio in the cytosolic fraction. The highest concentration of nitrotyrosine was found in the particulate fraction from the right ventricle after three days of hypoxia....

Microglia are the resident macrophage in the central nervous system (CNS) that actively survey their environment and participate in shaping neuronal circuits. Among the transcription factors necessary for microglia development, interferon regulatory factor 8 (IRF8) is a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. These studies suggest that lack of microglial IRF8 can subsequently impact neuronal function in disease, but the mechanisms underlying these effects remain unknown. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. To interrogate the impact of disrupted microglial IRF8 signaling on brain circuits, I first show by RNAseq that several genes known to regulate neuronal function are dysregulated basally in Irf8-/- brains. I then found that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to chemically-induced lethal seizures in Irf8-/- mice. Importantly, I also show that developmental synaptic pruning, a key function for microglia, proceeds normally in Irf8-/-mice. Finally, I identified that these IRF8-dependent effects on circuits are due to elevated TNF-α in the CNS as genetic or acute pharmacological blockade of TNF-α in the Irf8-/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. The next steps are to use cell-specific genetic approaches to manipulate this signaling, which I have further developed over the course of this project.

<p>  </p> <p>Osteoarthritis (OA) is prevalent in equine and can be career-ending for performance horses due to lameness limitations and decreased quality of life. OA is a progressive, multifactorial disease that compromises the synovial joints' normal function, resulting in subchondral bone and articular cartilage deterioration over time. OA is a complex disease that impacts the entire joint, wherein activation of the innate immune system has an essential role in the disease progression and the development of pain. The synovial membrane, or the synovium, is a crucial contributor to the inflammation of diseased joints, regardless of the intra-articular tissue type initially affected. Synoviocytes are a predominant cell type of the synovium and contribute to inflammation by releasing key mediators and degradative enzymes, such as interleukin (IL)-6, IL-1β, a disintegrin, and metalloproteinase (ADAM) domains, and matrix metalloproteinases (MMPs). The production of pro-inflammatory molecules sequentially influences the expression of degradative enzymes and cartilage destruction. Therefore, the pathophysiological processes within synovial joints afflicted by OA can be further understood by studying the characteristics of synoviocytes.</p> <p>We aimed to investigate the inflammatory component of OA in an <em>in vitro</em> model using a primary cell line of equine fibroblast-like synoviocytes (eqFLS) stimulated with tumor necrosis factor-alpha (TNF-α) to represent an initial inflammatory stimulus. Our studies have shown that stimulating eqFLS with TNF-α for 24 hours significantly increased the gene expression of pro-inflammatory biomarkers. Among several pro-inflammatory candidate genes assayed, only pro-inflammatory cytokine IL-6 gene expression could be detected reproducibly following stimulation with the TNF-α gene in eqFLS. We characterized the pro-inflammatory response of eqFLS and utilized this system to examine the impact of novel therapeutic compounds designed <em>in-silico</em> with the goal of reducing the inflammatory response of eqFLS. A piperazine-based compound (C3) and its derivative (02-09) were primarily designed to mimic the interactions of the growth factor pigment epithelium-derived factor (PEDF) with its receptor, the non-integrin laminin receptor 1 (LAMR1). Based on previous <em>in vitro</em> studies in the laboratory, C3 and 02-09 had been proposed to have a strong potential for inhibiting inflammation while reducing angiogenesis and chondrocyte hypertrophy. The efficacy of these two novel compounds on eqFLS was examined in the present work by assessing the gene expression levels of inflammatory biomarkers, including IL-6, IL-1β, IL-8, ADAMs, and MMPs relative to a control housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in various study designs. An <em>in-vitro</em> screen with the IL-1β promoter driving a reporter green fluorescent protein (GFP) was also designed to detect and track the inflammatory response of eqFLS by imaging following stimulation with or without (+/-) TNF-α relative to controls. This screen will be utilized in future studies to potentially identify more effective compounds in the LAMR1-interacting series. The current findings suggest that the novel compounds, especially 02-09, might exhibit an anti-inflammatory effect on eqFLS; therefore, it is a potential therapeutic agent in modulating inflammation during OA development. </p> <p><br></p>

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24h with LPS (100ng/ml) or LPS (100ng/ml)+IFNγ (1μg/ml). Cells were subsequently stimulated with LPS or HKSa (10μg/ml) for 24h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα (3070±711pg/ml and 217±9pg/ml, respectively) and IL-6 (237±8.9pg/ml and 56.2±2.9pg/ml, p<0.05, n=3, respectively) in control cells compared to basal levels (<25pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p<0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580pg/ml, p<0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076pg/ml, p<0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470pg/ml, p<0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor β1 (TGF-β1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-β1 on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-α, 10 ng/ml of TGF-β1, or combination, for 6 h. TNF-α exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-α upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-α treatment. In addition, caspase-3-like activity was significantly activated following TNF-α treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-α. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-α induced apoptosis. In contrast, the addition of TGF-β1 attenuated all of the aforementioned effects of TNF-α. Our results demonstrate that TGF-β1 can decrease TNF-α-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-α-induced caspase gene expression.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20533号 / 生博第375号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 米原 伸, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM