Research Activities
We would like to note that our lab’s policy is to respect the scientific interests and ideas of all students and scientists in forging their research projects. We believe that each project has conceptual novelty with the potential to broadly impact the worldwide scientific community.
(1) HMGB1 in the regulation of inflammation and oncogenesis
The activation of innate immune responses mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors in response to nucleic acids is crucial to protective and pathological immunity. We have found that high-mobility group box (HMGB) proteins function as universal sentinels for nucleic acids for these pathways. HMGBs bind to all immunogenic nucleic acids and show a positive correlation between the affinity of the interaction and the immunogenic potential of the response. We showed that HMGB proteins are required for the activation of all nucleic acid-sensing receptors, i.e., cytosolic nucleic-acid-sensing receptors, TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicated a hierarchy wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs (Nature, 462, 99-103, 2009).
Although the innate immune system can discriminate between many forms self and non-self, nucleic acids themselves are common to host and infectious microbes. Thus, recognition of self versus non-self nucleic acids by innate receptors must be in a delicate balance, and any imbalance of this system may be closely linked to autoimmune and auto-inflammatory disorders. Indeed, there is ample evidence for a connection between nucleic-acid-mediated immune responses in animal models of autoimmune diseases and human diseases such as SLE. Hence, designing inhibitors that impede nucleic-acid-mediated immune responses might be a useful strategy to treat autoimmune diseases. Towards this aim, we developed an oligodeoxynucleotide (ODN), termed ISM ODN that shows very high binding affinity to HMGB proteins and yet does not evoke innate immune responses. We found that ISM ODN treatment in mice suppresses the development of experimental autoimmune encephalomyelitis (EAE), an autoimmune demyelinating disease model of human multiple sclerosis, in which nucleic-acid-sensing TLRs contribute to the development of the disease. Interestingly, ISM ODN treatment also blocks LPS-mediated death by endotoxin shock (Proc Natl Acad Sci U S A,108,11542-7, 2011), suggesting that ISM ODN also blocks HMGB1-mediated inflammatory responses.
We also generated Hmgb1-floxed mice to achieve the conditional inactivation of the gene in a cell- and tissue-specific manner (Proc Natl Acad Sci U S A, 110, 20699-704, 2013). In these studies we observed that HMGB1, released by liver cells, is required for liver metastasis of tumor cells (unpublished). Thus, ISM ODN may have therapeutic potential for variety of disease conditions such as autoimmunity, sepsis and cancer metastasis. We are currently analyzing modifications of the extracellularly released HMGB1 to gain further mechanistic insight on how nuclear HMGB1 is released outside of a cell to evoke these varied immunoregulatory functions.
(2) Identification and characterization of self-derived molecules which regulate inflammation and immunity
Cellular components released into the external milieu as a result of cell death are generally termed damage-associated molecular patterns (DAMPs). Although DAMPs are conventionally thought to be protective to the host by evoking inflammatory responses, there is the prevailing notion that release of DAMPs can also underlie or exacerbate disease development. How resultant DAMP-mediated responses are regulated remained to be fully addressed. We identify prostaglandin E2 (PGE2) as a DAMP that negatively regulates immune responses. The production of PGE2 is augmented under cell death-inducing conditions via the transcriptional induction of the cyclooxygenase 2 (COX2) gene and that cell-released PGE2 suppresses the expression of genes associated with inflammation, thereby limiting the cell's immunostimulatory activities.
Consistent with this, inhibition of the PGE2 synthesis pathway potentiates the inflammation induced by dying cells. We also provided in vivo evidence for a protective role of PGE2 released upon acetaminophen-induced liver injury as well as a pathogenic role for PGE2 during tumor cell growth (Proc Natl Acad Sci U S A.,113, 3844-9, 2016). Our study thus placed this classically known lipid mediator in an unprecedented context-that is, an inhibitory DAMP vis-a-vis activating DAMPs, which may have translational implications. We have then identified several self-derived molecules, which we believe are the genuine activating DAMPs; more detailed analysis is underway.
(3) The IRF family of transcription factors in the regulation of inflammation and immunity
Interferon regulatory factors (IRFs) constitute a family of transcription factors that commonly share a novel helix-turn-helix DNA-binding motif. Following the initial identification of two structurally related members, IRF-1 and IRF-2 in our laboratory, seven additional members have been reported. Their functional roles, through interactions with their own or other members of the family of transcription factors, are becoming clearer in the regulation of host defense, such as innate and adaptive immune responses and oncogenesis (Ann. Rev. Immunol., Vol. 19, 623-655, 2001；Nature Rev. Immunol., Vol. 6, 644-659, 2006). By generating mice deficient in either of these transcription factor genes, we adduced evidence for the essential role of IRF3 and IRF7 for the Type I IFN gene induction by the activation of innate receptors (Nature, Vol. 434, 772-777, 2005, Nature, Vol. 434, 1035-1040, 2005). In collaboration with Tak Mak and colleagues, we also showed there is a critical contribution of IRF5 in the induction of inflammatory cytokine genes (Nature, Vol. 434, 243-249, 2005). We recently generated Irf3-floxed mice to achieve the conditional inactivation of the gene in a cell- and tissue-specific manner. The analysis of these mice is in progress.
(4) Bacteriophage and intestinal inflammation
The intestinal microbiome is a dynamic system of interactions between the host and resident flora of microbes. Although a fine balance and mutually beneficial relationship is maintained under physiological conditions, imbalance in the ecosystem underlies the development and/or aggravation of disease such as inflammatory bowel disease. Our lab is interested in bacteriophages, which can infect intestinal bacteria and affect immunogenic potential or recognition by the host immune system, thereby contributing to intestinal diseases. In this context, we have isolated a new bacteriophage that, upon infection to a commensal bacterium in the intestine, potentiates inflammatory responses. Work is in progress to elucidate the mechanisms by which the phage infection potentiates the bacterium's immunogenicity and how this relates to development of intestinal inflammatory conditions. This work is a collaboration with Dr. Makoto Kuroda in National Institute of Infectious Diseases and Drs. Yasunori Tanji and Kazuhiko Miyanaga of Tokyo Institute of Technology.
(5) Inter-organ regulation of gut commensal bacteria for intestinal immune homeostasis
The composition of the gut commensal microbiota reflects the co-evolution of host and microbes to achieve intestinal homeostasis that is mutually beneficial. There has been much attention on how intestinal microbiota contributes to the regulation of the gut immune system, since its dysregulation underlies the development of inflammatory diseases in the intestine and other organs.
We have recently discovered a unique example of how distant organ-derived immunomodulatory molecule critically shapes symbiotic status of intestinal bacteria. In particular, we have shown that surfactant protein D (SP-D), which to date has been exclusively studied for its role in the lung homeostasis is also produced in gallbladder and secreted into intestinal lumen. SP-D binds selectively to commensal bacteria, thereby interfering with their overgrowth to maintain symbiotic status in the intestine. Indeed, SP-D-deficient mice manifest intestinal dysbiosis, accompanied by an abnormal development of regulatory T cells, and are highly susceptible to the development of DSS-induced colitis. Thus, our study is revealing a unique inter-organ regulation of intestinal immune homeostasis by dynamic cross-talk between gallbladder and intestine via SP-D with clinical implications such as cholecystectomy. This work is a collaboration with Dr. Atsushi Kumanogoh of Osaka University and Drs. Masahisa Hattori and Wataru Suda of The University of Tokyo.
(6) Nucleic acids and autoimmunity
There is increasing evidence that strongly indicates that RNA and DNA sensors of the innate immune system are primary contributors to autoimmune disease pathogenesis. It remains, however, unclear which RNA and/or DNA mediate disease development. During a course of our collaboration with a pharmaceutical company to screen TLR antagonist, we identified a low molecular RNA that strongly activates the immune system via TLR7 and whose contribution to autoimmunity has been known.
As such, we are focusing on the role of this RNA and its mechanism of activation of TLR in cooperation with other host cell-derived factors. We are also analyzing the contribution of this RNA to autoimmunity by genetically engineered mice for this RNA and are aiming at development of a compound that interferes with the function of this RNA for therapeutic purpose.
(7) CLR family of innate immune receptors in the regulation of tumor growth and metastasis
The eradication of tumor cells requires communication to and signaling by cells of the immune system. While natural killer (NK) cells are essential tumor-killing effector cells of the innate immune system, little was known about whether or how other immune cells recognize tumor cells to assist NK cells. We found that Dectin-1, a C-type lectin receptor (CLR) family of innate receptors, expressed on dendritic cells and macrophages is critical to NK-mediated killing of tumor cells that express N-glycan structures at high levels. Receptor recognition of these tumor cells causes the activation of IRF5 and downstream gene induction for the full-blown tumoricidal activity of NK cells. Consistent with this, we found mice genetically deficient in either Dectin-1 or IRF5 to show exacerbated in vivo tumor growth (Elife ;3:e04177. doi: 10.7554/eLife.04177, 2014).
Further, we found that Dectin-2, another CLR family member, is critical for the suppression of liver metastasis of cancer cells. Dectin-2 functions in resident macrophages in the liver, known as Kupffer cells, to mediate the uptake and clearance of cancer cells. Interestingly, Kupffer cells are selectively endowed with Dectin-2-dependent phagocytotic activity, with neither bone marrow-derived macrophages nor alveolar macrophages able to mediate these effects. Concordantly, subcutaneous primary tumor growth and lung metastasis are not affected by the absence of Dectin-2. In addition, macrophage C-type lectin (MCL) that forms a complex with Dectin-2 also contributes to the suppression of liver metastasis, highlighting the hitherto poorly understood mechanism of Kupffer cell-mediated control of metastasis that is mediated by the CLR innate receptor family (Proc Natl Acad Sci U S A., 113, 4097-14102, 2016).
Additional studies are in progress to gain further mechanistic insights on the CLR family-mediated anti-tumor immune responses and to provide new means with which to control tumors by harnessing these receptors.
This work is a collaboration with Dr. Yuichiro Iwakura of Tokyo University of Science, Dr. Shinobu Saijyo of Chiba University and Dr. Sho Yamazaki of Kyusyu University.
