Planned Research Projects

A cellular society of inflammation for pulmonary fibrosis

Representative

Kouji Matsushima, MD, PhD

Professor, Division of Molecular Regulation of Inflammatory and Immune Diseases Research Institute for Biomedical Sciences Tokyo University of Science

http://www.prevent.m.u-tokyo.ac.jp/

Pulmonary fibrosis is a serious and fatal condition that is characterized by the excessive production of extracellular matrix leading to lung dysfunction. Pulmonary fibrosis is associated with a broad range of lung diseases including idiopathic pulmonary fibrosis and interstitial lung diseases such as those caused by exposure to environmental pollutants. Because pulmonary fibrosis is progressive, irreversible, and lacks effective therapies, there is a need for the development of novel early-stage diagnostic methods and preventive interventions. A key pathological feature of pulmonary fibrosis is the formation of “fibrotic lesions” in the lung interstitium that are composed of a diversity of cell types including fibroblasts, epithelial cells, endothelial cells, macrophages, neutrophils and lymphocytes. It is hypothesized that the progression of pulmonary fibrosis is regulated by a cell-cell interaction network formed between these cells (“a cellular society of inflammation”). However, the dynamics and underlying mechanisms of this network remain unclear.

Single-cell transcriptome technology is a new approach for comprehensive analysis of mRNA expression at the level of individual cells. We (Hashimoto et al.) have developed an inexpensive high-throughput microwell-based single-cell transcriptome method called Nx1-seq. Using this technology, we will individually analyze the diversity of cells present in fibrotic tissues comprehensively and at high resolution. We will collect data on the temporal and spatial changes that occur in the pathology of fibrosis and reconstruct the cellular interaction network at each analysis point. Based on this reconstructed network, we will generate a 4-dimensional spatio-temporal predictive model of the changes that occur as pulmonary fibrosis progresses from the earliest pathological stage. We will optimize this model though close collaboration with Topic A03 groups and though validation by experiments with genetically modified mice and methods such as single-molecule FISH. Using this model, we aim to predict the key turning points in the pathological process from initial lung injury through to the development of pulmonary fibrosis. Furthermore, by conducting interventional experiments targeting these points of regulation in the cellular society of inflammation, we aim to characterize the “pre-disease” fibrotic condition, identify early-stage diagnostic markers, and devise novel therapeutic strategies for pulmonary fibrosis. At the same time, we will use this model of the cellular society of inflammation to evaluate the environmental factors and molecular targets identified by Topic A02 collaborators. Through this research, we expect to open new avenues in preventive medicine that will bring society closer to conquering pulmonary fibrosis.

Elucidation of inflammation cellular society in liver cirrhosis

Representative

Shuichi Kaneko, MD, PhD

Professor, Department of system biology, graduate school pf advanced preventive medical sciences, Kanazawa University

Chronic infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) and alcohol abuse are major causes of liver cirrhosis. Nowadays, we can suppress HBV viral proliferation by antiviral drugs, nucleotide/nucleoside analogues, to stop progress to liver cirrhosis. And more, we can also eradicate HCV by direct antiviral agents, DAAs, to stop progress to liver cirrhosis.

In 21th century, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are rising issues as causes of liver cirrhosis and liver cancer. NAFLD/NASH is diagnosed in more than 10% of population and 20-30% of health check subjects and are markedly increasing internationally. NAFLD/NAS is closely related to diabetes mellitus (DM) and consequently progress to liver cirrhosis and liver cancer. Several diagnosis methods, preventive methods and treatment drugs based on exogenous and endogenous risk factors discovered from previous basic researches including genetic analysis on NAFLD/NASH have been tried to result in low efficacy and effectiveness. Therefore, construction of new health maintenance system for these diseases is urgent task for us.

We have analyzed pathogenesis of inflammation and fibrosis in NAFLD/NASH and reported first in the world that overnutrition affects environment in liver and changes nutrition metabolism pathway, and liver products Selenoprotein P (named “hepatokine” against “adipokine” produced from visceral fat) to worsen DM. Moreover, we have established mouse model in which only food causes NAFLD/NASH to progress to liver cirrhosis and liver cancer and have researched NAFLD/NASH developing new transgenic mice of new molecules which are important for liver inflammation and fibrosis such as Selenoprotein P, leukocyte cell-derived chemotaxin 2 (Lect2) and platelet -derived growth factor (PDGF)-c. We have also collected more than 100 clinical samples under ethical guidelines and started single cell transcriptome research.

Based on these research backgrounds, in this project, we collaborate with researchers in other areas to elucidate inflammation cellular society in liver cirrhosis. We plan to identify key cells which most closely related to progress to liver cirrhosis and obtain 3-dimentional information in the tissue consequently to establish 4-dimentional model. We concretely define simple fatty liver as pre-symptomatic state based on this model, and organize genetic information related to prevention of progression from NAFLD/NASH to liver cirrhosis. We also develop diagnostic markers for progress from simple fatty liver to NASH or from NAFLD/NASH to liver cirrhosis and preventive methods and treatments for NASH and liver cirrhosis.

Our NAFLD/NASH mouse models have high similarity to human clinical course and pathological findings because the models are established by food only without any chemicals. We elucidate inflammation cellular society in liver cirrhosis using our models.
We collect blood and liver samples with time from NAFLD/NASH mouse models to analyze comprehensive gene expression and clarify the relationship between gene expression and morphological changes such as inflammation and fibrosis or various chemical factors.

We analyze single cell transcriptome using samples from simple fatty liver as pre-symptomatic state, NASH with early inflammation, NASH with fibrosis and liver cirrhosis in cooperation with inflammation cellular society analysis center in coordination section of this project. Based on results from these analyses, we identify key cells for transition of inflammation cellular society consisting with hepatocytes, cholangiocytes, endothelial cells, hepatic satellite cells, Kupffer cells, infiltrating inflammatory cells and fibroblasts. After identification of key cells, we establish 4-dimentional simulation model including intercellular interaction network obtaining positional information of key cell based on tissue distribution of specific molecules in the cells.

We hybridize transgenic mice of important genes for liver inflammation and fibrosis (Selenoprotein P, Lect2, PDGF-c) with NAFLD/NASH mice. We validate importance of key cells and specific molecules for progression of NAFLD/NASH by analyses of genes related to liver inflammation and fibrosis in those hybrid mice.

Moreover, we conduct comprehensive gene expression analysis in each step, simple fatty liver as pre-symptomatic state, NAFLD/NASH with early inflammation, NASH with fibrosis and liver cirrhosis using human clinical samples. We validate established model in human by whole genome sequence in liver tissue comparing with that in lymphocytes in peripheral blood.

We research predictive markers for progress from simple fatty liver as pre-symptomatic state to NASH, diagnostic markers for progress of liver cirrhosis and target molecules to prevent those progresses which contribute to new drugs development in cooperation with other researchers and coordination section in this project.

Inflammation and related molecular preventive medicine in kidney diseases

Representative

Takashi Wada, MD, PhD

Professor, Kanazawa University

The number of the patients, who need chronic dialysis therapy is still increasing in Japan. Chronic kidney diseases(CKD) is a high risk not only for end stage renal disease but also for life-threatening cardiovascular diseases. Moreover, these risks become higher in advanced grade of CKD. Therefore, the prevention of kidney disease may improve mortality and morbidity in CKD patients.

Chronic inflammation plays an important role for the progression of kidney diseases. We have reported the contribution of inflammation to various kinds of kidney diseases, such as acute kidney injury, glomerulonephritis and lupus nephritis. Even in diabetic kidney disease, chronic inflammation accelerates the kidney injury. The regulation of inflammatory cells/molecules improves progressive kidney diseases in rodent models. These facts suggest the control of chronic inflammation could be a therapeutic target for progressive kidney diseases.

In the project, we focus on the chronic inflammation, especially microenvironment inflammation in kidney diseases. We hypothesize that inflammatory microenvironment networks may lead to disease onset and escalation in kidney diseases. We think that understanding of inflammation cellular society could be the key for the new approach for prevention of progressive kidney disease.

Elucidation of Molecular Basis of Inflammation and Fibrosis Regulated by Quantity and Quality of Organ Lipids in Inflammation Cellular Society and Development of New Preventive Strategy for Inflammatory Diseases

Representative

Hitoshi Shimano, MD PhD

Professor, Department of Endocrinology and Metabolism, Faculty of Medicine, University of Tsukuba

Quantity and quality aspects of organ lipids play important roles in the pathophysiology of chronic inflammation, fibrosis, and organo-pathy. SREBP-1 and CREBH are transcription factors that regulate quantity of lipids in cells and organs towards accumulation and depletion, respectively. Elovl6 is a member of fatty acid elongases that regulate quality of organ lipids. These factors are likely to play crucial roles in the development and progression of inflammatory and presumably fibrotic diseases.

Using single cell transcriptome analysis and lipidomics in cell and mouse models, we will attempt to elucidate the molecular basis of SREBP-1/CREBH/Elovl6 involvement in inflammation cellular society and develop new preventive strategy for inflammatory diseases.

We are going to analyze

1. the importance and molecular mechanisms of SREBP-1/CREBH/Elovl6 on the chronic inflammation and fibrosis in metabolic syndrome
2. exactly what kinds of lipids are important for the prevention of inflammation and fibrosis in metabolic diseases
3. the role of lipid metabolism in myofibroblast.

Environmental stress and biological response: Epigenome and Proteome analysis

Representative

Seiichiroh OHSAKO, DVM, PhD

Associate professor, The University of Tokyo, Graduate School of Medicine, Laboratory of Environmental Health Science

http://www.cdbim.m.u-tokyo.ac.jp/english/research/05.php

Chronic inflammation is caused by various environmental stresses, and exposure to chemicals including electrophiles, such as methylmercury and acrylamide. Such environmental pollutants and industrial chemicals causes not only carcinogenesis, but also central nervous system abnormalities and lead to cognitive impairment even when ingested with extremely small amounts. However, the detailed mechanism of pathogenesis is hardly elucidated. Chronic inflammation of the central nervous system may be closely involved. We hypothesize that the impaired autophagy and inflammasome caused by electrophile activated Nrf2 may play an important role in neuronal degeneration or suppression of noradrenaline production. We have also reported that continuous exposure to a low-level chemical causes susceptible property for cancer by experimental animal models. This alteration is due to epigenetic changes including DNA demethylation. We have found that activation of Ahr, a receptor for exogenous chemicals, actively demethylates DNA using the DNA repair pathway called as BER resulting in long term memory of epigenome. From the standpoint of preventive and/or preemptive medicine, it is extremely important to elucidate the pathogenesis induced by environmental factors and to clarify the molecular mechanism underlying premature state of disease. In this research team, we will focus on the cognitive impairment, which is particularly important in recent years, and try to define premature state leading the chronic inflammation in the central nervous system associated with the environmental stress response. For this aim, we will integrate various omics data obtained by affordable methylome (MSD-AFLP) and proteome analysis, as well as with those using by comprehensive single cell transcriptome (Nx1-seq) which will be performed by the supervising team. Finally we will develop prediction method for premature state of disease, eventually by integrative mathematical analysis.

Investigation on perturbed signals in inflammatory cell society attributed to RNF213 mutations in the development of vascular stenotic/occlusive legions

Representative

Akio Koizumi, M.D., Ph.D.

Professor, Department of Health and Environmental Sciences, Kyoto University Graduate School of Medicine

http://hes.med.kyoto-u.ac.jp/index-e.html

Moyamoya disease (MMD) is an arteriopathy characterized by stenotic/occlusive lesions primarily in the circle of Willis but also in other systemic arteries. We identified RNF213 as a susceptible gene and have functionally characterized RNF213. We also found that a mutation, p.R4810K, is a founder mutation common to Japanese, Korean and Chinese patients and its carrier population accounts for 2-3 % in Japanese or Korean and 0.8% in Chinese with 15 million in East Asian population. Since its penetrance is known to be enigmatically low (1/150), environmental factors including stimuli, which elicit chronic inflammation, have been speculated to induce MMD with RNF213 R4810K. We have demonstrated that angiogenesis response after hypoxia exposure in the cerebrum is lowered by R4810K mutation. As such, RNF213 may likely induce premature arterial lesions toword stenosis or occlusive lesions.　 However, its detailed pathological process, organ specificity and interaction with other molecules, or signal pathways involved remain unknown.

In this research group, we will conduct single cell transcriptome to elucidate the RNF213 R4810K enigma in collaboration with the Inflammatory Cell Society Analyzing Center. With this collaboration, we can reveal trancriptome profile in adaptation process of inflammatory cell population (i.e., premature disease stage) under stress: In this analysis we will consider arterial endothelial cells and arterial smooth muscle cells as major constituents in the inflammatory cell society. With these analyses, we will reconstruct big data information in 3 or 4 dimensional space in collaboration with Dr. Ikeo, facilitating to understand pathological process through time space and organ specificity. With those results, we will reconstruct a model in which the perturbation is brought by RNF213 R4810K mutation: the model will be confirmed by genetic modified mice.

It has been hypothesized that inflammation, which is induced by the interaction between environmental stimuli and susceptible genes, causes the vascular legions. In this research project, the mode and mechanism of the interaction in the axis of RNF213 will be elucidated. With such results, effective prevention strategy will be established. In conclusion, we will aim to elucidate molecular mechanisms of vascular stenosis covering from atherosclerosis to vascular stenosis in general. Our results can contribute to development of a new scientific area focusing on gene-and-environmental interaction.

Development of molecular-targeting prevention of inflammatory diseases with chemical biology

Representative

Toshiyuki Sakai, M.D., Ph.D.,

Professor, Department of Drug Discovery Medicine, Kyoto Prefectural University of Medicine

Various functional food factors and chemopreventive agents activate the tumor suppressor gene product RB by inducing CDK inhibitors such as p15, p16, and p21. In consequence, since the proliferation of cancer cells is inhibited, it is very useful for molecular-targeting prevention of cancer to activate RB. However, it is likely that the activation of RB also induces cellular senescence and senescence-associated secretory phenotype (SASP) followed by perturbation of inflammation cellular society, resulting in elicitation of chronic inflammatory diseases and progression of cancer. In the present research, we focused on induction of SASP which is a potential weak point of RB activation. As shown in the lower figure, since induction of SASP is regulated by not only RB but also the molecules regulating NF-κB such as mTOR, p38, insulin, and SIRT1, we perform a comprehensive screening for SASP inhibitors. On the other hand, since NK cells eliminate the cells causing SASP, we also perform a screening for the compounds which activate NK cells. These screening methods are developed based on our original cell-based assay, by which we developed the first-in-class MEK inhibitor trametinib. Furthermore, we identify the direct binding proteins of hit compounds by our chemical biology method and discover inflammation-modulating target molecules which are the target proteins accounting for SASP inhibition by hit compounds. We then elucidate the novel mechanisms of inflammation governed by these inflammation-modulating target molecules and develop more effective molecular-targeting prevention of cancer and chronic inflammatory diseases by combinations of SASP inhibitors and RB activators.

Development and data analysis of information method for cell sociology based on single cell sequence data.

Representative

Kazuho Ikeo, PhD

Associate Professor, National Institute of Genetics

In order to elucidate the formation process of inflammatory memory as intercellular interaction at the cellular or molecular level, qualitative and quantitative information on constituent cells of inflamed tissue composed of thousands of cells is collected as “cell state variable”. We will develop the methods necessary for accumulation and integration and actual data analysis using it. We aim to make it possible to describe a cellular society as “a place of inflammation” based on a single cell profile. For that purpose, we build a model that correlates the interactions to the various state changes observed in inflamed tissues and describes the “memory of the field” as a state change of cells and cell populations. In addition, we aim to establish the necessary method and construct the analysis flow for that. In cooperation with other teams, the obtained results verify the correctness of the model by observing whether or not predictable results can be obtained from the above model. Through this process, we aim to understand “memory of the place”.