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Center for Experimental Medicine and Systems Biology

Laboratory of Innate Immunity
自然免疫研究分野
Professor

Kensuke Miyake, M.D., Ph.D.

教授

医学博士

三宅健介

Pathogen sensors, such as Toll-like receptor (TLR), play sentinel roles in detecting
pathogenic ligands during infection and induce both innate and acquired immune
responses. Meanwhile, excessive TLR responses are strongly associated with fatal
diseases such as septic shock and autoimmune diseases. For this reason, immune
system must strictly control TLR responses to avoid disruption of homeostasis.
However, molecular mechanisms involved in TLR regulation are not fully elucidated.
We have previously shown that TLRs are regulated by various TLR associating
molecules including MD-2, PRAT4A and Unc93B1. Our goal is to uncover molecularmechanism that is indispensable for appropriate TLR responses using geneticallyengineered mice.

1.  Targeting the nucleic acids-sensing TLRs fortherapeutic intervention in autoimmune diseases
Yuji Motoi1, Ryutaro Fukui1, Takuma Shibata1,Kensuke Miyake1,2
1Division of Innate Immunity, Department of Microbiology and Immunology, 2Laboratory of Innate Immunity, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The
University of Tokyo, 4-6-1 Shirokanedai, Minatoku,
TOKYO1208-8639, Japan.
TLR7 senses microbial-derived RNA in endolysosome, but can also erroneously respond to self-derived RNA. In fact, it has been reported that TLR7-dependent signaling promote autoimmune diseases.
Thus, TLR7 can be therapeutic target. Although antibodies (Abs) are powerful tools for therapeutic intervention, TLR7 has been excluded from targets for
Ab-mediated intervention because of its lack of cellsurface expression. Despite this expectation, wefound an anti-TLR7 Ab dose-dependently inhibits
TLR7 responses in dendritic cells, macrophages and Bcells. For this reason, we evaluated the therapeutic effect of anti-TLR7 Ab in Unc93b1D34A/D34A mice thatcause thrombocytopenia, splenomegaly and chronic
active hepatitis due to TLR7 hyper-responsiveness,and found that thrombocytopenia in Unc93b1D34A/D34A
mice was significantly improved by the treatment
with anti-TLR7 mAb. Furthermore, splenomegaly
and hepatitis in mice treated with the anti-TLR7 mAb
were also significantly remedy compared with control antibody.
On basis of these results, we established anti-human TLR7 Ab for blocking human TLR7 responses invitro. Moreover, we generated human TLR7 transgenic (huTLR7 Tg) mice. We plan to use HuTLR7 Tg
mice to evaluate the effects of anti-human TLR7 Ab invivo.
In addition, TLR8 also recognize mouse TLR7 ligands in human and is involved in exacerbation ofRheumatoid Arthritis. Thus, in case of human disease, the anti-human TLR8 Ab that inhibits human
TLR8 responses might work in clinical application.
For this reason, we also constructed both anti-human
TLR8 Abs and human TLR8 transgenic mice to verify
our hypothesis.

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2.  Microbiome ssRNA as an environmental cue toactivate TLR13-dependent tissue-protective
programs in CD5Lhi hepatic macrophages
Ryota Sato1, Kaiwen Liu1, Takuma Shibata1, Katsuaki Hoshino2, 3, Kiyoshi Yamaguchi4, Toru Miyazaki5, Ryosuke Hiranuma1, Ryutaro Fukui1, YujiMotoi1, Yuri Fukuda-Ohta6, Yun Zhang1, Tatjana
Reuter7, Yuko Ishida8, Toshikazu Kondo8, Tomoki
Chiba9, Hiroshi Asahara9, 10, Masato Taoka11, Yoshio
Yamauchi11, Toshiaki Isobe11, Tsuneyasu Kaisho3, 6,
Yoichi Furukawa4, Eicke Latz7, 12, Kensuke Miyake1*1
Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo; Minato-ku,
Tokyo 108-8639, Japan. 2 Department of Immunology, Faculty of Medicine, Kagawa University, Miki,Kagawa 761-0793, Japan. 3 Laboratory for Inflammatory Regulation, RIKEN Center for Integrative
Medical Science (IMS-RCAI), Yokohama, Kanagawa 230-0045, Japan. 4 Division of Clinical GenomeResearch, The Institute of Medical Science, The
University of Tokyo; Minato-ku, Tokyo 108-8639,Japan.
5
The Institute for AIM Medicine, Tokyo 162-8666,
Japan. 6 Department of Immunology, Institute ofAdvanced Medicine, Wakayama Medical University, Kimiidera, Wakayama 641-8509, Japan. 7 Institute of Innate Immunity, University Hospital Bonn,
University of Bonn, 53127 Bonn, Germany. 8 Department of Forensic Medicine, Wakayama MedicalUniversity, Kimiidera, Wakayama 641-8509, Japan.
9Department of Systems Biomedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 1138501, Japan. 10 Department of Molecular and Experimental Medicine, The Scripps Research Institute,La Jolla, CA 92037, USA.
11
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University; Tokyo 1920397, Japan. 12 Deutsches Rheuma Forschungszentrum Berlin (DRFZ), 10117 Berlin, Germany.
Hepatic macrophages maintain liver homeostasis,but little is known about the signals that activate thehepatoprotective programs within macrophages.
Here, we show that toll-like receptor 13 (TLR13), asensor of bacterial 23S ribosomal RNA (rRNA), sensesmicrobiome RNAs to drive tissue-protective responses in CD5Lhi hepatic macrophages. Splenomegaly andhepatomegaly developed in the absence of the endosomal RNase, RNaseT2, via TLR13-dependent macrophage proliferation. Furthermore, TLR13 in hepatic
Ly6Clo macrophages activated the transcription factors LXRα and MafB, leading to expression of tissue-clearance molecules, such as CD5L, C1qb, andAxl. Consequently, Rnaset2‒/‒ mice developed resistance to acute liver injury caused by challenges withacetaminophen and lipopolysaccharide + D-galactosamine. TLR13 responses in Rnaset2‒/‒ mice wereimpaired by antibiotics, suggesting that TLR13 wereactivated by microbiome rRNAs, which was detectedin the sera and hepatic macrophages. Repeated administration of wild-type mice with the TLR13 ligand,rather than other TLR ligands, selectively increased
the number of Kupffer cells, which expressed immunoregulatory and tissue-clearance genes as hepatic
macrophages in Rnaset2‒/‒ mice did. Our results suggest that microbiome ssRNA serves as an environmental cue for initiating tissue-protective TLR13 responses in hepatic macrophages.
3.  Aberrant monocytopoiesis drives granuloma
development in sarcoidosis
Ryosuke Hiranuma 1, Ryota Sato 1, Kiyoshi Yamaguchi 2, Satoshi Nakamizo 3,4, Kenichi Asano5, Takuma
Shibata 1, Ryutaro Fukui 1, Yoichi Furukawa 2,Kenji
Kabashima 3,6, Kensuke Miyake 1,81
Division of Innate Immunity, Department of Microbiology and Immunology, 2 Division of Clinical Genome Research, The Institute of Medical Science,
The University of Tokyo, Japan. 3Department ofDermatology, Kyoto University Graduate School ofMedicine, Kyoto, Japan. 4Alliance Laboratory for
Advanced Medical Research, Kyoto University
Graduate School of Medicine, Kyoto, Japan. 5Laboratory of Immune Regulation, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences,
Tokyo 192-0392, Japan. 6A*STAR Skin ResearchLabs (A*SRL), Agency for Science, Technology, andResearch (A*STAR), 8A Biomedical Grove, IMMUNOS Building, Biopolis, Singapore.
In sarcoidosis, granulomas develop in multiple organs including the liver and lungs. Although mTORC1
activation in macrophages drives granuloma development in sarcoidosis by enhancing macrophage proliferation, little is known about the macrophage subsets that proliferate and mature into granulomamacrophages. Here, we show that aberrantly increased monocytopoiesis gives rise to granulomas ina sarcoidosis model, in which Tsc2, a negative regulator of mTORC1, is conditionally deleted in CSF1R-expressing macrophages (Tsc2csf1rΔ mice). In Tsc2csf1rΔ mice, common myeloid progenitors (CMPs),
granulocyte-monocyte progenitors (GMPs), common
monocyte progenitors/monocyte progenitors (cMoPs/MPs), inducible monocyte progenitors (iMoPs), andLy6Cint CX3CR1low CD14- immature monocytes(iMOs), but not monocyte-dendritic cell progenitors(MDPs) and common dendritic cell progenitors(CDPs), accumulated and proliferated in the spleen.
Consistent with this, monocytes, neutrophils, andneutrophil-like monocytes increased in the spleens of
Tsc2csf1rΔ mice, whereas dendritic cells did not. The
adoptive transfer of splenic iMOs into wild-type micegave rise to granulomas in the liver and lungs. In
these target organs, iMOs matured into Ly6Chi classi-

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cal monocytes/macrophages (cMOs). Giant macrophages (gMAs) also accumulated in the liver andlungs, which were similar to granuloma macrophages
in expression of cell surface markers such as MerTKand SLAMF7. Furthermore, the gMA-specific genes
were expressed in human macrophages from sarcoidosis skin lesions. These results suggest thatmTORC1 drives granuloma development by promoting the proliferation of monocyte/neutrophil progenitors and iMOs predominantly in the spleen, and thatproliferating iMOs mature into cMOs and then gMAs
to give rise to granuloma after migration into the liverand lungs in sarcoidosis.

Publications
Hiranuma R, Sato R, Yamaguchi K, Nakamizo S, Asano K, Shibata T, Fukui R, Furukawa Y, Kabashima
K, Miyake K. Aberrant monocytopoiesis drives
granuloma development in sarcoidosis. Int Immunol. dxad054. doi: 10.1093/intimm/dxad054. 2023.
Liu G, Haw TJ, Starkey MR, Philp AM, Pavlidis S, Nalkurthi C, Nair PM, Gomez HM, Hanish I, Hsu AC,
Hortle E, Pickles S, Rojas-Quintero J, Estepar RSJ,Marshall JE, Kim RY, Collison AM, Mattes J, Idrees
S, Faiz A, Hansbro NG, Fukui R, Murakami Y,Cheng HS, Tan NS, Chotirmall SH, Horvat JC, Foster PS, Oliver BG, Polverino F, Ieni A, Monaco F,Caramori G, Sohal SS, Bracke KR, Wark PA, Adcock
IM, Miyake K, Sin DD, Hansbro PM. TLR7 promotes smoke-induced experimental lung damage
through the activity of mast cell tryptase. Nat Commun. 14(1):7349. doi:10.1038/s41467-023-42913-z.
2023

Chowdhury SI, Inui M, Yamazaki T, Tomono S, Takagi H, Biswas M, Saitoh SI, Miyake K, Akashi-Takamura S. The anti-TLR4 monoclonal antibody Sa1521 enhances inflammatory cytokine production inLPS-stimulated macrophages. FEBS Lett. 597(9):1246-1260. doi: 10.1002/1873-3468.14619. 2023
Kuchitsu Y, Mukai K, Uematsu R, Takaada Y, Shinojima A, Shindo R, Shoji T, Hamano S, Ogawa E, Sato
R, Miyake K, Kato A, Kawaguchi Y, Nishitani-Isa
M, Izawa K, Nishikomori R, Yasumi T, Suzuki T,Dohmae N, Uemura T, Barber GN, Arai H, Waguri
S, Taguchi T. STING signalling is terminatedthrough ESCRT-dependent microautophagy ofvesicles originating from recycling endosomes. Nat
Cell Biol. 25(3):453-466. doi: 10.1038/s41556-02301098-9. 2023