運動誘発性炎症とリポ多糖による炎症に対するポリフェノールの抗炎症作用の解析

早大学位記番号:新7712早稲田大

A New Pathogenesis of Albuminuria: Role of Transcytosis

Transcytosis is an important intracellular transport process by which multicellular organisms selectively move cargoes from apical to basolateral membranes without disrupting cellular homeostasis. In kidney, macromolecular components in the serum, such as albumin, low-density lipoprotein and immunoglobulins, pass through the glomerular filtration barrier (GFB) and proximal tubular cells (PTCs) by transcytosis. Protein transcytosis plays a vital role in the pathology of albuminuria, which causes progressive destruction of the GFB structure and function. However, the pathophysiological consequences of protein transcytosis in the kidney remain largely unknown. This article summarizes recent researches on the regulation of albumin transcytosis across the GFB and PTCs in both physiological and pathological conditions. Understanding the mechanism of albumin transcytosis may reveal potential therapeutic targets for prevention or alleviation of the pathological consequences of albuminuria

THE ROLE OF HIGH MOBILITY GROUP BOX-1 PATHOBIOLOGY IN ANGIOTENSIN II-INDUCED ABDOMINAL AORTIC ANEURYSMS

Abdominal aortic aneurysms (AAAs) are permanent luminal dilations of the vessel wall that can result in rupture and death. There is currently no evidence-based treatment to prevent or attenuate the development of this devastating condition. Although vascular inflammation is known to be one of the hallmarks of AAA, underlying mechanisms that initiate inflammatory pathways in the aorta are not clearly known. High-mobility group box 1 (HMGB1), a highly conserved nonhistone DNA-binding nuclear protein, may contribute to vascular diseases. Since whole-body genetic deletion of HMGB1 is embryonic lethal, pharmacological approaches have been used to manipulate HMGB1 in mice. However, it remains desirable to genetically manipulate HMGB1 to further understand its role. In the body of work presented here, we assessed the contributing role of HMGB1 in AAA development and the efficacy of neutralization and a novel antisense oligonucleotides (ASOs) approach to deplete HMGB1 in mice. To examine the role of HMGB1 in AAA development, we assessed the gene expression profile of human and murine aneurysmal samples, indicating a marked upregulation of inflammatory pathways and HMGB1 in the abdominal aorta of diseased tissue. Further validations at the early stages of experimental AAA by utilizing the Angiotensin II (AngII) model also indicated a marked increase of HMGB1 protein abundance in the abdominal aorta of male LDLr-/- mice. To determine the role of HMGB1 inhibition in AngII-induced AAAs, a monoclonal anti-HMGB1 antibody (2G7) or an isotype-matched control were intraperitoneally injected into 8-10-week-old male LDLr-/- mice that were infused with AngII for 28 days. Neutralizing HMGB1 with a low dose did not show a significant decrease in the abdominal aortic diameter of AngII-infused mice. Next, we examined the efficacy of seven different ASOs in reducing HMGB1 protein abundance at selected intervals. Either ASOs or phosphate-buffered saline (PBS) were injected into male C57BL/6J mice (8-10-week-old) at days 0 and 3 in the initial week and then once a week during the remainder of the study. Subsequently, mRNA and protein abundance of HMGB1 were determined in the various tissues. After screening various ASOs to determine the most optimum version and to further investigate the role of systemic HMGB1 inhibition in aneurysm formation, hypercortisolemic male mice fed a Western diet were infused with AngII for four weeks to induce aneurysm and were injected with PBS or HMGB1 ASO. Our results indicated a profound significant attenuation of AAA in ASO administered group. Collectively, our data established that the ASO approach could significantly decrease HMGB1 expression, and its inhibition in an experimental aneurysm model can profoundly attenuate the AngII-induced AAA formation. Further, utilizing an ASO approach to inhibit HMGB1 can provide more clear insights into understanding the biological functions of HMGB1 with strong clinical significance

Bench-to-bedside review: High-mobility group box 1 and critical illness

High-mobility group box 1 (HMGB1) is a DNA-binding protein that also exhibits proinflammatory cytokine-like activity. HMGB1 is passively released by necrotic cells and also is actively secreted by immunostimulated macrophages, dendritic cells, and enterocytes. Although circulating HMGB1 levels are increased relative to healthy controls in patients with infections and severe sepsis, plasma or serum HMGB1 concentrations do not discriminate reliably between infected and uninfected critically ill patients. Nevertheless, administration of drugs that block HMGB1 secretion or of anti-HMGB1 neutralizing antibodies has been shown to ameliorate organ dysfunction and/or improve survival in numerous animal models of critical illness. Because HMGB1 tends to be released relatively late in the inflammatory response (at least in animal models of endotoxemia or sepsis), this protein is an attractive target for the development of new therapeutic agents for the treatment of patients with various forms of critical illness

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

This is the publisher's version, also available electronically from http://www.jci.org/articles/view/18704While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation

Mechanisms of Methylglyoxal-elicited Leukocyte Recruitment

Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In hyperglycemic conditions, an increased MG level has been linked to the development of diabetes and the accompanying vascular inflammation encountered at both macro- and microvascular levels. The present study explores the mechanisms of MG-induced leukocyte recruitment in mouse cremasteric microvasculature. Biochemical and intravital microscopy studies performed suggest that administration of MG (25 and 50 mg/kg) to mouse cremaster muscle tissue induces dose-dependent leukocyte recruitment in cremasteric vasculature with 84-92% recruited cells being neutrophils. MG treatment up-regulated the expression of endothelial cell (EC) adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) via the activation of nuclear factor-κB (NF-κB) signalling pathway and contributed to the increased leukocyte rolling flux, reduced leukocyte rolling velocity, and increased leukocyte adhesion, respectively. The inhibition of NF-κB blunted MG-induced endothelial adhesion molecule expression and thus attenuated leukocyte recruitment. Further study of signalling pathways revealed that MG induced Akt-regulated transient glycogen synthase kinase 3 (GSK3) activation in ECs, which was responsible for NF-κB activation at early time-points (< 1 h). After MG activation for 1 h, the endothelial GSK3 activity was decreased due to the up-regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1), which was responsible for maintaining NF-κB activity at later time-points. Silencing GSK3 or SGK1 attenuated P-selectin, E-selectin and ICAM-1 expression in ECs, and abated MG-induced leukocyte recruitment. SGK1 also promoted cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activity which was partially involved in ICAM-1 expression. Silencing CREB blunted ICAM-1 expression while P-selectin and E-selectin levels remained unaffected. MG also induced GSK3 activation in isolated neutrophils after 30 min treatment, an effect that was not responsible for MG-elicited Mac-1 expression. These data suggest the sequential activation of GSK3 and SGK1 in ECs as the pivotal signalling mechanism in MG-elicited leukocyte recruitment. Additionally, MG-treatment led to uncoupling of endothelial nitric oxide synthase (eNOS) following MG-induced superoxide generation in ECs. MG triggered eNOS uncoupling and hypophosphorylation associated with superoxide generation and biopterin depletion in EA.hy926 ECs. In cremaster muscle, as well as in cultured murine and human primary ECs, MG increased eNOS monomerization and decreased 5,6,7,8-tetrahydroboipterin (BH4)/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by NG-nitro-L-arginine methyl ester (L-NAME) or 5,6,7,8-tetrahydroneopterin (NH4). These observations confirm that MG administration triggers eNOS uncoupling. In murine cremaster muscle, MG triggered the reduction of leukocyte rolling velocity and the increases in rolling flux, adhesion, emigration and microvascular permeability. MG-induced leukocyte recruitment was significantly attenuated by supplementation of BH4 or sepiapterin or suppression of superoxide by L-NAME confirming the role of eNOS uncoupling in MG-elicited leukocyte recruitment. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary ECs, suggesting the impaired BH4 biosynthesis caused by MG. Taken together, these data suggest that vascular inflammation and endothelial dysfunction occurring in diabetes may be linked to GSK3/SGK1 regulated adhesion molecule expression, as well as the uncoupling of eNOS evoked by elevated levels of MG. These findings not only provide a better understanding of the role of MG in the development of diabetic vascular inflammation, but also suggest the potential therapeutic targets for MG-sensitive endothelial dysfunction in diabetes

Dual cytokine inducing properties of HMGB1

High mobility group box protein 1 (HMGB1) is a nuclear protein that can be released either passively by necrotic cells or actively by stimulated cells. Extracellular HMGB1 is a potent inducer of inflammation and the importance of HMGB1 as a mediator in a number of inflammatory diseases including rheumatoid arthritis, sepsis and ischemia‐reperfusion injury, has been demonstrated by successfully targeting the protein in preclinical models. The aim of my thesis was to characterise the cytokine‐inducing properties of HMGB1 and to study which receptors were required for cytokine induction. Several studies have indicated that HMGB1 can co‐operate with other pro‐ inflammatory molecules to induce inflammation. To further study this mechanism we formed complexes of HMGB1 together with the exogenous TLR ligands LPS, Pam3CSK4 and CpG‐ODN or the endogenous ligands IL‐1α and IL‐1β. Stimulation of macrophages or synovial fibroblasts with these different HMGB1 complexes resulted in significantly enhanced cytokine production as compared to stimulation with each ligand alone (papers I and II). Importantly, HMGB1 selectively enhanced the stimulatory activity of certain molecules as it did not display this activity with all tested ligands. In papers II and III the receptor requirements of HMGB1 complexes were studied. HMGB1 in complex with LPS, Pam3CSK4 and IL‐ 1α/β stimulated cytokine release via the TLR4, TLR2 and IL‐1RI receptors, respectively, demonstrating that cytokine induction by HMGB1 complexes is dependent on the receptor for the respective partner molecule. In paper IV we demonstrated that cytokine release stimulated by uncomplexed HMGB1 was dependent on TLR4 but not on RAGE or TLR2, and that a direct association of HMGB1 and TLR4 was detected both in vitro and in vivo. Using site‐ directed mutagenesis we furthermore determined that the cysteine in position 106 of HMGB1 was required for both binding to TLR4 and for cytokine induction. In summary, in this thesis I have demonstrated that HMGB1 has the ability to induce cytokine production in two ways: through forming complexes with certain danger molecules and thereby increasing their stimulatory activities, and through direct interaction with the TLR4 receptor. The TLR4‐mediated endogenous cytokine‐inducing capacity of HMGB1 requires a cysteine in position 106, while the enhancing capacity of HMGB1‐partner ligand complexes is independent of HMGB1 ligation to TLR4 but dependent on signalling via the partner molecule receptor. Neither mechanism involved an interaction of HMGB1 with its suggested receptor RAGE. These results are of value for designing HMGB1‐targeting therapies that focus on blocking only certain HMGB1 functions or certain receptor interaction

RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS: EXPRESSION AND SIGNALING

The NF-B transcription factor family plays a central role in many aspects of the immune response, and activation of this family of transcription factors has been shown to trigger many disease processes. Thus, the ability to modulate NF-kB activity may be an attractive way to treat these diseases. We used an in vitro cell-based assay to test potential NF-kB inhibitors by measuring their effect on IL-1-induced expression of the NF-B dependent intracellular adhesion molecule-1 (ICAM-1, CD54). To develop the cell-based system we sorted IL-1b-responsive U373 human astrocytoma cells to obtain a population of cells with minimum background expression and maximum induced expression of CD54 following stimulation with IL-1. We tested ethyl pyruvate, a novel anti-inflammatory drug candidate, and the ability of related compounds to block activation of NF-kB activity by measuring the expression of CD54 on U373 cells exposed to IL-1. 4-hydroxyphenylpyruvic acid was the best inhibitor of CD54 upregulation. We further tested the compounds using the mouse macrophage-like RAW 264.7 cell line which produce a variety of cytokines and nitric oxide (NO•) following exposure to lipopolysaccharide (LPS) in an NF-kB-dependent manner. The drugs downregulated LPS-induced IL-6 production, iNOS upregulation, and NO• production following the same efficacy trend observed in the primary screening using CD54 expression in U373 cells. These studies show the ease of using an endogenous reporter gene (i.e., CD54) and FACS analysis to rapidly characterize the relative efficacy of pharmacologic inhibitors. A second completely unrelated topic of the dissertation dealt with the receptor for advanced glycation end-products (RAGE). RAGE is thought to be important in a variety of pathological conditions, including diabetes, sepsis, atherosclerosis, renal diseases, hypertension and Alzheimer's disease. However, RAGE proximal signaling events are still unclear. We were able to establish that original RAGE, sequenced from bovine lung, is only present in the lung. This observation was based on antibody specificity, Northern blotting and N-glycosylation analysis. One of the antibodies that we used (H-300, Santa Cruz, CA) was very selective for lung RAGE and not cross-react with other RAGE isoforms. Only lung RAGE had a transcript size of 1.4 kb as determined by Northern blot and only lung RAGE was N-glycosylated. Non-lung tissues and cell lines appeared to express their own unique RAGE isoforms. Non-lung derived cell lines were permissive for lung RAGE isoform expression but lung derived cell lines were not. Interestingly, all transfected cell lines (of lung and non-lung origin) expressed RAGE mRNA transcripts. In addition, we established that previously described endogenous soluble RAGE (esRAGE) does not contain any of the canonical RAGE epitopes, but includes sequence encoded in intron 9. RAGE knockout mice lose esRAGE isoform along with the canonical one confirming that esRAGE originates from the RAGE gene. Signaling studies with pro-inflammatory stimuli in mouse lung slices of wild-type and knockout mice revealed the importance of RAGE in LPS and IL-1-induced inflammatory response, but not when reported RAGE ligands, including AGEs, HMGB1 and S100B, were applied

Effects of diabetes on microglial physiology: a systematic review of in vitro, preclinical and clinical studies

Diabetes mellitus is a heterogeneous chronic metabolic disorder characterized by the presence of hyperglycemia, commonly preceded by a prediabetic state. The excess of blood glucose can damage multiple organs, including the brain. In fact, cognitive decline and dementia are increasingly being recognized as important comorbidities of diabetes. Despite the largely consistent link between diabetes and dementia, the underlying causes of neurodegeneration in diabetic patients remain to be elucidated. A common factor for almost all neurological disorders is neuroinflammation, a complex inflammatory process in the central nervous system for the most part orchestrated by microglial cells, the main representatives of the immune system in the brain. In this context, our research question aimed to understand how diabetes affects brain and/or retinal microglia physiology. We conducted a systematic search in PubMed and Web of Science to identify research items addressing the effects of diabetes on microglial phenotypic modulation, including critical neuroinflammatory mediators and their pathways. The literature search yielded 1327 records, including 18 patents. Based on the title and abstracts, 830 papers were screened from which 250 primary research papers met the eligibility criteria (original research articles with patients or with a strict diabetes model without comorbidities, that included direct data about microglia in the brain or retina), and 17 additional research papers were included through forward and backward citations, resulting in a total of 267 primary research articles included in the scoping systematic review. We reviewed all primary publications investigating the effects of diabetes and/or its main pathophysiological traits on microglia, including in vitro studies, preclinical models of diabetes and clinical studies on diabetic patients. Although a strict classification of microglia remains elusive given their capacity to adapt to the environment and their morphological, ultrastructural and molecular dynamism, diabetes modulates microglial phenotypic states, triggering specific responses that include upregulation of activity markers (such as Iba1, CD11b, CD68, MHC-II and F4/80), morphological shift to amoeboid shape, secretion of a wide variety of cytokines and chemokines, metabolic reprogramming and generalized increase of oxidative stress. Pathways commonly activated by diabetes-related conditions include NF-κB, NLRP3 inflammasome, fractalkine/CX3CR1, MAPKs, AGEs/RAGE and Akt/mTOR. Altogether, the detailed portrait of complex interactions between diabetes and microglia physiology presented here can be regarded as an important starting point for future research focused on the microglia–metabolism interface.30 página