論文の公開元へSingle-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction
概要
Title
Single-cell transcriptomics of human
cholesteatoma identifies an activin A-producing
osteoclastogenic fibroblast subset inducing bone
destruction
Author(s)
Shimizu, Kotaro; Kikuta, Junichi; Ohta, Yumi et
al.
Citation
Nature Communications. 2023, 14(1), p. 4417
Version Type VoR
URL
rights
https://hdl.handle.net/11094/93150
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
Article
https://doi.org/10.1038/s41467-023-40094-3
Single-cell transcriptomics of human
cholesteatoma identifies an activin
A-producing osteoclastogenic fibroblast
subset inducing bone destruction
Received: 20 November 2022
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Accepted: 12 July 2023
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Kotaro Shimizu 1,2,3, Junichi Kikuta 1,2,4 , Yumi Ohta3, Yutaka Uchida
Yu Miyamoto1,2, Akito Morimoto1,2, Shinya Yari 1,2, Takashi Sato3,
Takefumi Kamakura3, Kazuo Oshima3, Ryusuke Imai3, Yu-Chen Liu 5,6,
Daisuke Okuzaki 5,6, Tetsuya Hara7, Daisuke Motooka5,6, Noriaki Emoto
Hidenori Inohara3 & Masaru Ishii 1,2,4
1,2
,
7
,
Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing
loss and brain abscess formation. However, the pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a singlecell RNA sequencing analysis on human cholesteatoma samples and identify a
pathogenic fibroblast subset characterized by abundant expression of inhibin
βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes
osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in
these fibroblasts results in decreased osteoclast differentiation in a murine
model of cholesteatoma. Moreover, follistatin, an antagonist of activin A,
reduces osteoclastogenesis and resultant bone erosion in cholesteatoma.
Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone
destruction via the induction of local osteoclastogenesis, suggesting a
potential therapeutic target.
Cholesteatoma is a type of chronic middle ear inflammation that
expands with bone erosion, destroying temporal bone structures and
causing symptoms such as hearing loss, dizziness, facial paralysis, and
meningitis1. Furthermore, cholesteatoma constitutes an epidermal
cyst that arises from the epithelial layer of the tympanic membrane
and is composed of three layers: matrix, composed of the keratinized
stratified squamous epithelium; perimatrix, the surrounding layer of
the matrix that contacts temporal bone and contains collagen fibers,
fibroblasts, and endothelial cells; and cystic components that form the
most internal layer, containing keratin debris and necrotic tissue shed
from the matrix2. Multiple mechanisms have been proposed to explain
bone erosion in cholesteatoma, including osteoclast activation3,4, acid
1
Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. 2WPIImmunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. 3Department of Otorhinolaryngology-Head and Neck Surgery,
Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. 4Laboratory of Bioimaging and Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan. 5Genome Information Research Center, Research Institute for Microbial Diseases,
Osaka University, Suita, Osaka 565-0871, Japan. 6Laboratory of Human Immunology (Single Cell Genomics), WPI-Immunology Frontier Research Center,
Osaka University, Suita, Osaka 565-0871, Japan. 7Laboratory of Clinical Pharmaceutical Science, Kobe Pharmaceutical University, Higashinada, Kobe 658e-mail: jkikuta@icb.med.osaka-u.ac.jp; mishii@icb.med.osaka-u.ac.jp
8558, Japan.
Nature Communications | (2023)14:4417
1
Article
lysis5, pressure necrosis3,6, inflammatory mediators7–9, enzymatic
mediators10,11, and combinations of ≥2 of these mechanisms. Nevertheless, the mechanism that underlies local bone destruction in cholesteatoma has not been elucidated. The only effective treatment at
present is complete surgical excision, but the rate of postoperative
recurrence remains unsatisfactory12.
A previous study showed that numerous osteoclasts were
observed on the eroded bone surfaces adjacent to cholesteatomas,
compared to unaffected areas, and that fibroblasts in the cholesteatoma perimatrix express receptor activator of NF-κB ligand (RANKL), a
protein essential for osteoclast differentiation and function4,13.
Because multiple subtypes of fibroblasts have been reported in other
inflammatory diseases, such as rheumatoid arthritis14, it is possible that
cholesteatoma also contains several subtypes of fibroblasts. However,
previous studies have not provided an overview of cell types and
subsets present in cholesteatoma. Here, we performed single-cell RNA
sequencing (scRNA-seq) analysis of human cholesteatoma specimens
to clarify the contributions of these cells to bone destruction in cholesteatoma. The results showed that cholesteatoma perimatrix fibroblasts express high levels of activin A, a secreted protein that acts in
cooperation with RANKL to induce mature osteoclast formation. We
conducted a detailed assessment of the relationship between activin A
Fig. 1 | scRNA-seq analysis of human cholesteatoma and skin specimens.
a Representative gating strategies used in cholesteatoma and skin samples. Live
(calcein+ AAD−) CD45− cells. Scale bars: 5 mm. b UMAP plot of scRNA-seq data from
19,273 cells labeled by sample condition. Samples were obtained from three pairs
Nature Communications | (2023)14:4417
https://doi.org/10.1038/s41467-023-40094-3
and bone erosion in cholesteatoma, and the results showed that activin
A is a potential therapeutic target for cholesteatoma.
Results
Single-cell RNA sequencing analysis identified a cholesteatomaspecific pathogenic fibroblast subset
To elucidate the mechanism underlying bone erosion in cholesteatoma, we performed scRNA-seq analysis using human cholesteatoma
tissues surgically resected from patients. As there are no “normal”
tissues in the middle ear that correspond to cholesteatoma, retroauricular skin at the incision site was used as a control for cholesteatoma. Previously, we reported the involvement of fibroblasts in bone
erosion in cholesteatoma mediated via RANKL signaling4; therefore,
we focused on pathogenic nonimmune cells, such as fibroblasts or
keratinocytes, in the present study. Both cholesteatoma and control
skin specimens were enzymatically digested and sorted into CD45– live
cell populations (Fig. 1a). Then, we applied scRNA-seq to the collected
cells and analyzed the data sets. We first carried out quality control to
exclude poor-quality data; we obtained a reliable data set consisting of
8357 cells from the cholesteatoma samples and 10,916 cells from the
control skin samples obtained from three patients. Next, we performed clustering analysis and visualized the results in uniform
of cholesteatoma and control skin samples labeled according to sample condition.
c UMAP plot of scRNA-seq data labeled according to cell type identified in PanglaoDB. The main cell types were keratinocytes, fibroblasts, and endothelial cells.
2
Article
https://doi.org/10.1038/s41467-023-40094-3
Fig. 2 | Identification of cholesteatoma fibroblasts and INHBA upregulation in
cholesteatoma fibroblasts. a The proportions of sample conditions in each cluster
identified by scRNA-seq. The proportion of cholesteatoma varied among clusters.
Clusters consisting of cholesteatoma alone were considered cholesteatomaspecific clusters. b Proportion of patients in each cluster identified by scRNA-seq.
The proportion of patients varied among clusters. Clusters consisting of one
patient were considered clusters with large sample biases. c Genes upregulated in
cholesteatoma fibroblasts compared to control fibroblasts. Genes with top 10 zscores are shown in order of fold change. d INHBA upregulation in cholesteatoma
fibroblast clusters.
manifold approximation and projection (UMAP). As shown in Fig. 1b,
both cholesteatoma and control dermal cells showed several clusters
(blue: cholesteatoma; orange: control skin; Supplementary Fig. S1
shows color coding by the patient). Our clustering method classified
the samples into 17 clusters, and we annotated the cell types based on
marker gene expression (Fig. 1c). We proposed that the data sets
consisted of 11 distinct cell types, including keratinocytes, fibroblasts,
endothelial cells, and osteoclasts (Fig. 1c; Supplementary Figs. S2 and
S3). Because keratinocytes, fibroblasts, and endothelial cells were
major populations in the data sets, subsequent analysis focused on
these cells.
To identify cholesteatoma-specific keratinocytes, fibroblasts,
and endothelial cell clusters, we evaluated the proportions of
cholesteatoma-derived cells and normal dermal cells in each cluster.
The keratinocytes in cluster 1 and fibroblasts in cluster 5 were strongly
biased toward the cholesteatoma samples, whereas other clusters,
including endothelial cell clusters 0 and 7, were unbiased (Fig. 2a). We
also examined bias between patients in keratinocyte cluster 1 and
fibroblast cluster 5. The cell proportion in fibroblast cluster 5 was
unbiased toward any particular patient, whereas keratinocyte cluster 1
was strongly biased toward patient 1 (Fig. 2b). We also confirmed that
cluster 5 in fibroblasts had higher condition specificity and lower
sample bias (Supplementary Fig. S4a–c). Taken together, our data
suggest that cluster 5 represented a cholesteatoma-specific pathogenic fibroblast subset. ...
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