Publicly Offered Research Projects (FY2020-)

Inflammation Cell Society in cardiac homeostasis and pathobiology

Ichiro Manabe, MD, PhD

Department of Disease Biology and Molecular Medicine, Chiba University

We have reported that macrophages and fibroblasts play divergent and essential roles in the maintenance of homeostasis as well as pathobiology. In particular, macrophages can assume diverse phenotypes and functions in response to environmental cues. We and others have found that the changes of macrophages depend on the dynamic regulation of epigenome. In this study, we aim to analyze the dynamic changes in the transcriptome and epigenome at a single cell level in macrophages and fibroblasts in the heart. We will analyze the communication among the cells to elucidate the dynamism of the inflammation cellular society in stress response and heart failure.

Regulation of inflammation cellular sociology by lipids toward better understanding of allergic diseases

Makoto Murakami, PhD

Laboratory of Microenvironmental Metabolic Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo

Transepidermal invasion of environmental factors, such as bacterial toxins and harmful antigens, due to skin barrier dysfunction can trigger excessive activation of type 2 immunity, leading to development of atopic dermatitis, a refractory chronic skin disease, and even rhinitis and asthma in distal organs, known as a process of “allergic march”. Therefore, appropriate control of the skin barrier is an urgent task in considering the prevention of allergic diseases. Quantitative and qualitative changes in lipids have great influences on the inflammatory cell society. The purpose of this study is to unveil the molecular mechanism underlying the process of transitioning from skin homeostasis (undiseased) to its failure (prolonged) and further to chronic allergic condition (irreversible) with lipid as a keyword, and accordingly, to create a new concept concerning the molecular preventive control of allergy based on the maintenance of lipid microenvironment. By taking advantages of a series of gene-manipulated mice for various phospholipase A2s in combination with comprehensive lipidomics and single cell transcriptome, we herein aim to clarify the roles of particular lipid pathways in the regulation of allergy, especially focusing on immune responses of epidermal origin and controls of mast cell microenvironment.

Elucidation of the inflammatory cell network in functional alteration of hair follicle stem cells

Toshiaki Ohteki

Professor, Medical Research Institute, Tokyo Medical and Dental University

http://www.tmd.ac.jp/mri/bre/index.html

Hair is generated from hair follicle stem cells (HFSCs) in the bulge region, where HFSCs are usually protected, as an immune privilege area, from adverse events. On the other hand, chronic IFN signal is involved in human alopecia (alopecia areata and scarring alopecia), but the specific role of IFN signal remains unknown.
Using mouse models showing chronic IFN signal-dependent hair loss, e.g. Irf2^-/- mice and IMQ-painted WT mice, we will elucidate the detailed hair loss mechanism induced by chronic inflammation from the viewpoint of the functional alteration of HFSC by interacting with immune cells. In this context, Irf2^-/- mice show IFN signal- and CD8⁺T cells-dependent hair loss with aging, but we found that this hair loss was not caused by direct CD8⁺T cell-mediated cytotoxicity (unpublished). Based on the background, in this year, we analyze the results of comprehensive gene expression in the immune cells and HFSCs of both models using “ligand-receptor database”, and rank the ligands expressed by immune cells and the receptors on HFSCs. In the second year, we will examine the role of predicted ligand/receptor signals in the functional alteration of HFSCs. Using a hair removal experiment that can analyze hair regeneration ability of HFSCs, we will inject a candidate ligand into the skin of control WT mice and an Ab blocking the candidate ligand/receptor interaction into Irf2^-/- mice and IMQ-painted WT mice. The importance of candidate ligand/receptor signals is evaluated by using the inhibition or recovery of hair regrowth as an index. Furthermore, we will examine whether the hair loss is improved in Irf2^-/- mice lacking the candidate ligand/receptor. From these studies, we will elucidate the roles of chronic IFN signals on HFSC alteration and hair loss.

Control of the Intestinal Immune System through Innate Immune Receptors and Its Effects on the Systemic Immune System

Representative

Yoichiro Iwakura, D. Sc.

Professor and Director, Center for Experimental Animal Models, Research Institute for Biomedical Sciences, Tokyo University of Science

http://www.rs.tus.ac.jp/iwakuralab/

Our intestinal tract is colonized by many types of microbiota. The number and diversity of these microbes are always changing in any given moment throughout our life, and the majority of these organisms are not harmful invaders but cohabitants living with us as their one-and-only home. Indeed, our metabolic and immune functions are largely dependent on our intestinal microflora. By activating host antimicrobial innate immune mechanisms and by directing the development of adequate adaptive immune cells of the mucosal and systemic immune system, commensal microbes contribute to the host defense against pathogens and host immune homeostasis.
Recent studies have revealed the importance of pattern recognition receptors (PRRs), which recognize pathogen- associated molecular patterns, in the host defense against pathogens and intestinal immune homeostasis. C-type lectin receptors (CLRs) is a group of such PRRs and bind carbohydrate structures on pathogens, such as fungi and mycobacteria, in a Ca²⁺-dependent manner, providing an important mechanism for the protection against these pathogens. Dectin-1 is one of type II CLRs that contains a carbohydrate recognition domain in its extracellular carboxyl terminus and an immunoreceptor tyrosine-based activation motif in its intracellular amino terminus, and highly expressed in dendritic cells, macrophages (Mfs), and neutrophils. Previously, the group of Gordon Brown and we showed that Dectin-1 is the receptor for β-1, 3- linked glucans with β-1, 6-linked branches (β-glucans) and plays an important role for the host defense against fungi by producing Th17 cytokines and reactive oxygen species (Saijo et al., Nat. Immunol., 2007). We have also shown that Dectin-2 is the receptor for α-mannans and important for the host defense against fungal infection (Saijo et al., Immunity, 2010). Because Dectin-1 and Dectin-2 are expressed in the colonic lamina propria (cLP) and Peyer’s patches in the small intestine and β-glucans and α-mannans are contained in many foods such as mushrooms, yeasts, and seaweeds, we investigated the role of these CLRs in the intestinal immune system.
Recently, we reported that Dectin-1 deficiency causes an increase of a specific lactobacillus species (Lactobacillus murinus) in the colon because of a decrease of a group of anti-microbial proteins, and suppresses the development of colitis by promoting regulatory T-cell (Treg) expansion in a lactobacillus-dependent manner (Tang et al., Cell Host & Microbe, 2015). Furthermore, we found that IL-17F is preferentially induced by Dectin-1 signaling (Kamiya et al., Mucosal Immunol., 2018), and induces antimicrobial proteins such as phospholipase A2 and suppresses growth of Treg-inducing bacteria, Clostridium cluster XIVa as well as L. murinus. Thus, inhibition of IL-17F, but not IL-17A, signaling induce Treg cell expansion in the colon resulting in the suppression of colitis (Tang et al., Nat. Immunol., 2018). In this project, we will analyze the roles of C-type lectins in the regulation of systemic immune system and in the development of tumors.

A cellular society for inflammation and regeneration of skeletal muscle

Yumiko Oishi

Professor, Department of Biochemistry and Molecular Biology, Nippon Medical School

Sarcopenia, age-related skeletal muscle loss partly due to insufficient muscle regeneration, is the leading cause of functional decline and metabolic dysregulation of the elderly. While chronic inflammation impairs muscle regeneration, insufficient muscle regeneration promotes chronic inflammation and tissue fibrosis. Indeed, the inflammatory signal is essential for the muscle stem cell activation and muscle regeneration, however, it is still unclear how damage-induced inflammation and regeneration is balanced. In this project, I will elucidate the mechanism of cellular network (inflammation cellular society) that govern a series or inflammation, regeneration and muscle tissue repair by combining single cell transcriptome analysis and global transcriptional/epigenetic analyses. In particular, I will focus on the communication between satellite cell (muscle-specific stem cell), fibroblast, and macrophages to clarify the mechanism of homeostatic stress response and their role in pathogenesis of sarcopenia.

Dynamic assessment of Th/Treg imbalance in inflammatory bowel disease pathogenesis

Takashi Sekiya, Ph.D.

Chief, Department of Immune Regulation, National Center for Global Health and Medicine

Uncontrolled helper T (Th) cell differentiation and attenuated regulatory T (Treg) cell induction is a major cause of inflammatory bowel disease (IBD) pathogenesis. However, it is still largely unknown how such Th/Treg imbalance occur during the course of IBD progression. A caveat that prevent the molecular and cellular investigation of IBD pathogenesis is the lack of powerful experimental tools that can detect ongoing Th and Treg activation and differentiation during the course of IBD progression.
On the other hand, we have been investigating molecular mechanisms of Th/Treg cell differentiation, by focusing on roles of ‘Nr4a’ family of nuclear orphan receptors. To further develop the study, we have established a novel reporter mouse for Nr4a1 expression, that enable the detection of activated CD4+ T cells both in vitro and in vivo (Fig.1A). By utilizing this novel tool, we will analyze several important Th/Treg differentiation events in IBD pathogenesis, including: 1. sites, 2.cell subsets, 3.molecule, 4. microbiota, which play important roles in Th/Treg cell differentiation. We also check the potential of the molecules, cell subsets, and microbiota found in the above study as therapeutic agents for IBD (Fig.1B). Collectively, we are trying to reveal the mechanisms for dysregulated differentiation of Th/Treg cells, giving a hint to the development of a novel therapeutic method for IBD.

Novel regulatory mechanism of lung fibrosis by amino acid transporter SLC15A3

Toshihiko Kobayashi

Research Institute, National Center for Global Health and Medicine Deputy Project Director

Fibrosis is a severe medical condition associated with collagen deposition in tissues and leading to the organ failure due to irreversible change. Among fibrosis in various organs, idiopathic pulmonary fibrosis (IPF) is one of intractable diseases still possessing high unmet medical needs. IPF affects approximately 3 million people worldwide. Steroids and anti-fibrotic drugs such as Nintedanib and Pirfenidone are applied as first-line agent, while they have serious problems of side effects and high medical costs. Therefore, identification of novel drug target and development of new therapeutic strategies are extremely important and urgent.
We recently found critical contribution of a lysosome-resident amino acid transporter SLC15A3 to the exacerbation of lung fibrosis. It is of note that, although the severity of bleomycin (BLM)-induced acute inflammation in SLC15A3 KO mice appears comparable to that in WT mice, at later phase inflammatory and pro-fibrotic parameters improved more than WT, resulting in faster recovery from fibrosis in KO mice. Thus, we speculate that SLC15A3 can be one of the key molecules that can direct the fate of inflammatory responses.
In this research project, we aim to clarify the inflammatory cell network centered on SLC15A3, which is composed of both hematopoietic and non-hematopoietic cells, and to comprehensively understand the conversion of the ‘inflammatory cell society’ by SLC15A3-deficiency in the lung tissue. Based on the results, we further examine the possibility that targeting SLC15A3 for fibrosis treatment.

Molecular insight into senescence in vivo by single-cell RNA-seq analysis

Yoshikazu Johmura

Assistant Professor, Institute of Medical Science, University of Tokyo

The most important problem to be solved of modern science in Japan is to extend healthy lifespan. Reorganization of the medical system and improvement of lifestyle and diet is clearly important to prolong our healthy lifespan. However, comprehensive understanding of the mechanism for aging and development of new technology for preventing age-associated disorders and deterioration of tissues and organs is necessary for the drastic improvement of healthy lifespan. Recent reports indicate that accumulation of senescent cells (p16-positive cells) in body accelerates age-related changes such as arteriosclerosis and kidney injury and limits healthy lifespan using genetically engineered mouse models. An important hallmark of senescent cells is the inability to proliferate in response to physiological mitotic stimuli, which can limit the ability of stem cells to renew tissues and organs. Another hallmark of senescence is the appearance senescence-associated secretory phenotypes (SASP), such as robust secretion of numerous growth factors, cytokines, proteases and other proteins, which can cause deleterious effects on the tissue microenvironment. What extent these two hallmarks contribute to aging and the related disorders remains elusive. Moreover, how senescent cells affect the gene expression patterns and epigenome of surrounding cells in vivo remains largely unknown. We succeeded to establish new mouse models for high-sensitive detection of senescent cells or conditional induction of cellular senescence and identify a novel drug for eliminating senescent cells (senolysis) in the first half of this research area. This study will apply these mouse models to comprehensive single-cell gene expression analysis for the understanding of the mechanisms by which senescence-induced inflammation contributes to aging and the related disorders. Moreover, effects of a novel senolysis drug on aging and the related disorders will be assessed by comprehensive single-cell gene expression analysis. Therefore, this study will be expected to contribute to the main purpose of ‘Inflammation Cellular Society’ and develop the preventions for aging and the related disorders.

Inflammation Cellular Sociology in cell death–induced chronic inflammation

Takayoshi Suganami, MD, PhD

Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University

http://www.riem.nagoya-u.ac.jp/en/meneki_kei.html

Accumulating evidence indicates that cell death triggers sterile inflammation and impaired clearance of dead cells causes non-resolving inflammation, however the underlying mechanisms are still unclear. We have reported that a novel cell death sensor, Mincle, plays critical roles in the pathophysiology of obesity-induced adipose tissue inflammation and acute kidney injury. Mincle is exclusively expressed in the macrophages forming “crown-like structure (CLS)”, in which dead parenchymal cells are surrounded and phagocytosed by macrophages. In this study, we aim to understand the role of Mincle in Inflammation Cellular Sociology in cell death–induced chronic inflammation. We will characterize Mincle-expressing macrophages, evaluate the spaciotemporal distribution of CLS, and analyze the dynamic changes in the transcriptome at a single cell level in cell death–induced chronic inflammation in the adipose tissue and the kidney.

Metabolic and epigenetic regulation of the cellular society of chronic inflammation

Representative

Masakatsu Yamashita, PhD

Professor, Department of Immunology, Graduate School of Medicine, Ehime University

Asthma and chronic obstructive pulmonary disease (COPD) are usually clinically distinct and have different causal mechanisms. In some elderly patients, there are features of both diseases, and this has been termed asthma-COPD overlap syndrome (ACOS). While nationwide aging has been advancing rapidly in Japan, the number of ACOS patient may rapidly increase. However, an affective treating method has not been established, and the onset mechanisms of ACOS remain to be elucidated.
We found that T cell-specific Bach2-deficient mice spontaneous developed ACOS-like chronic lung inflammation accompanying infiltration of eosinophils, neutrophils and lymphocytes. Increased innate-type Th2 cell response in Bach2-deficient lung CD4 T cells plays an important role in the onset of ACOS-like lung inflammation. Augmented activation of IL-7- and IL-33-mediated signals by Bach2 deficiency seem to be involved in the activation of innate-type Th2 cell response (Figure 1). We found that IL-33R-positive lung CD4 T cell express PD-1 and CD69. Furthermore,IL-7-dependet reprograming of cellular energy metabolism and subsequent epigenetic changes are required for the acquisition of innate-type Th2 function.
In this research project, we would like to reveal the metabolic and epigenetic regulation of the ACOS to clarify the regulatory mechanism of the cellular society of chronic lung inflammation.

Figure 1. Decreased Bach2 expression confers innate
Th2 function to CD4 T cells

Mechanism of postmenopausal NASH-HCC development and its preventive strategies based on the “Inflammation Cellular Sociology”

Yoshihiro Ogawa

Professor, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University
Professor, Department of Molecular and Cellular Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University

The metabolic syndrome is a cluster of visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia, and blood pressure elevation, which all increase independently a risk of atherosclerotic diseases Nonalcoholic fatty liver disease (NAFLD) is defined as excessive lipid accumulation in the liver and considered as the hepatic manifestation of the metabolic syndrome. It includes a spectrum of liver conditions ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) with advanced fibrosis and cirrhosis, which may occasionally cause hepatocellular carcinoma (HCC). Currently, NAFLD is an increasingly important cause of chronic liver disease in western countries, affecting more than 30% of the general population. The risk of NAFLD is gradually increased in men before the age of menopause, while in women, it increases abruptly after menopause, with increased risk of HCC in postmenopausal women.
Using melanocortin-4 receptor (MC4R)-deficient mice fed western diet (WD), we have developed a mouse model for human NASH, which exhibit obesity, liver steatosis, steatohepatitis, hepatic fibrosis, and eventually hepatocellular carcinoma. We found the unique histological features, which we have called hepatic crown-like structures (hCLS), in the NASH-like liver, where dead hepatocytes with large lipid droplet are surrounded by macrophages, and suggested that hCLS serves as an origin of hepatic inflammation and fibrosis during the progression from NAFL to NASH (Am. J. Pathol. 179: 2454-2463, 2011; PLoS ONE 8: e82163, 2013; JCI Insight 2: e92902, 2017). On the other hand, estrogen is known to suppress hepatic inflammation and fibrosis in the liver. However, how estrogen deficiency is involved in the development of NASH-HCC in postmenopausal women has not been fully elucidated.
In this study, using the ovariectomized MC4R-KO mice fed WD, we develop “postmenopausal NASH-HCC mice” and examine how hepatic inflammation and fibrosis occurs as a result of complex interaction among parenchymal hepatocytes and stromal cells such as macrophages within hCLS during estrogen deficiency. Accordingly, we aim to elucidate the entire picture of “Inflammation Cellular Sociology”, from the reversible pre-symptomatic state (NAFL), beyond the so-called “point of no return” state, to the irreversible diseased state (NASH-HCC) during the postmenopausal period.

Inflammation Cellular Sociology for the prevention of neurodegeneration

Takashi Shichita

Project Leader, Stroke Renaissance Project, Tokyo Metropolitan Institute of Medical Science

In the aging society, stroke and dementia are the major cause of shortening healthy life expectancy; however, few therapeutic methods have been established for improving neurological deficits and cognitive impairment. Inflammation has been recently demonstrated to be implicated in the progression of the pathologies in stroke and dementia. We have clarified the detailed molecular mechanisms underlying the trigger, prolongation, and resolution of cerebral post-ischemic inflammation. In the ischemic brain, endogenous inflammatogenic molecules (called damage-associated molecular patterns: DAMPs) which are released from a large number of necrotic brain cells activate infiltrating immune cells, leading to the formation of inflammation cellular sociology. Then, inflammatory factors are removed from the infarct region through the scavenger receptors expressed by macrophages and microglia. These macrophages and microglia also produce neurotrophic factors for neural repair; therefore, they are apparently reparative immune cells and are derived from the immune cells that compose the inflammation cellular sociology. In this study, we will clarify the detailed molecular mechanisms underlying the induction of reparative cells which are necessary for neural repair and the prevention of neuronal injuries in stroke and dementia.

Molecular mechanisms of sterile inflammation in Drosophila melanogaster

Sterile inflammation, or the activation of innate immunity without infectious stimuli, is thought to underlie lifestyle-related diseases and autoimmune diseases. However, the induction mechanisms of sterile inflammation is largely unknown. We previously reported that in Drosophila larvae, pinching by forceps, which possibly induces minor injury, can potentiate strong humoral innate immune responses. We showed that the expression of antimicrobial peptides (AMPs) induced by pinching is microbe-independent, and signaling pathways involved are largely independent from known innate immune signaling. In order to clarify molecular mechanisms of pinching-induced AMP expression, we have performed semi-genome wide RNAi screening and identified an important role of a chromatin remodeling factor.
In this study, we further investigate the molecular function of the factor in sterile innate immunity in Drosophila larvae, and reveal conserved mechanisms that are related to deleterious inflammation which leads a vast array of disorders and conditions in humans.

Fig. 1: Investigation of molecular mechanisms of sterile induction of innate immunity using Drosophila model

Study of vagal regulation of hepatic inflammation in response to nutritional changes

The changes in nutritional environment, including hormonal and nutrient changes, result in organ inflammation not only directly, but also indirectly via the brain and autonomic nerves. Especially, the vagal nerve regulates hepatic inflammation, which affects various hepatic function, for example, metabolism and regeneration, to adjust the liver to the nutritional environment. While the nutritional changes induce the vagal nerve activity fluctuation, under the condition of excessive nutrition, the vagal nerve loses this nutrient-dependent activity fluctuation and can be kept in a relatively inactivated condition. Because the activated vagal nerve suppresses hepatic inflammation, the loss of the vagal activity by excessive nutrition can lead to the onset and exacerbation of hepatic chronic inflammation, namely non-alcoholic steatohepatitis (NASH). Indeed, it has been reported that hepatic vagotomy results in hepatic chronic inflammation and we have also found that NASH is exacerbates by the absence of alpha-7 nicotinic acetylcholine receptor (A7AchR) in mice, which is a known effector of vagal-dependent regulation of inflammation. However, except the A7AchR, the role of the vagal nerve in hepatic inflammation remains to be fully elucidated. In this study, we investigate the role and mechanism of the vagal nerve in hepatic inflammation regulation.

Development of integrated analysis method of single-cell transcriptome data for elucidating inflammatory process

Representative

Hideo Matsuda, PhD

Professor, Osaka University, Graduate School of Information Science and Technology

In this study, we aim to elucidate the inflammatory and disease progression processes by integrating the analysis of heterogeneity of cell population and that of their inflammatory responses by single-cell transcriptome.

Specifically, we develop a method for inferring cell types based on the single-cell transcriptomic data with cell type labels in the ImmGen and GEO databases as positive examples. Furthermore, we develop a method to infer cell trajectories by ordering cell groups with their gene-expression similarities at various stages of inflammation. In addition, for human and mouse cell atlases including various types of transcriptomic data, we also proceed with the development of methods for removing batch effects that hinder the comparison and integration of the data, and for associating the data with biological imaging data. By combining the above methods with each other, we aim to implement integrated analysis of the progression of inflammatory responses and diseases.

Network Analysis Model and Efficient Methods for Single Cell Sequence Data

Early-stage preventive interventions are considered important to deal with chronic inflammatory diseases. One of the effective ways is probably to focus on “pre-disease” state before abnormalities of cell organisms are colonized as the inflammatory memory, although the pre-disease state has not been elucidated sufficiently. In order to elucidate the mechanism of the pre-disease, it would be desired to model information about gene mutation, epigenome alteration, and cell interaction as a “cellular society network” among cells, genes and molecules. We expect to establish a method for analyzing the propagation process of the inflammatory memory based on this model. However, conventional network model for comparing two states is not sufficient for analyzing a mechanism including three states, that is, normal, pre-disease, and inflammatory. Therefore, we will try the following two tasks:

• (1) Establishing a model of information propagation on a cellular society network containing the three states.
• (2) Developing efficient analysis methods for large-scale networks generated from single cell sequence data.