SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis
Takatoshi Enomoto, et al.
Takatoshi Enomoto, et al.
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Research Article Pulmonology

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis

Abstract

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

Authors

Takatoshi Enomoto, Yuya Shirai, Yoshito Takeda, Ryuya Edahiro, Shigeyuki Shichino, Mana Nakayama, Miho Takahashi-Itoh, Yoshimi Noda, Yuichi Adachi, Takahiro Kawasaki, Taro Koba, Yu Futami, Moto Yaga, Yuki Hosono, Hanako Yoshimura, Saori Amiya, Reina Hara, Makoto Yamamoto, Daisuke Nakatsubo, Yasuhiko Suga, Maiko Naito, Kentaro Masuhiro, Haruhiko Hirata, Kota Iwahori, Izumi Nagatomo, Kotaro Miyake, Shohei Koyama, Kiyoharu Fukushima, Takayuki Shiroyama, Yujiro Naito, Shinji Futami, Yayoi Natsume-Kitatani, Satoshi Nojima, Masahiro Yanagawa, Yasushi Shintani, Mari Nogami-Itoh, Kenji Mizuguchi, Jun Adachi, Takeshi Tomonaga, Yoshikazu Inoue, Atsushi Kumanogoh

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Figure 6

Spatial and longitudinal dynamics of SFTPB during the development of bleomycin-induced lung fibrosis.

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Spatial and longitudinal dynamics of SFTPB during the development of ble...
(A) Schematic representation of the experimental protocol used for Western blotting analysis of lung tissues, serum, serum EVs, and BALF from bleomycin-induced pulmonary fibrosis model mice and representative hematoxylin-eosin staining of lung tissues. (B–E) Western blotting analysis of SFTPB in lung tissues, serum, serum EVs, and BALF and quantification of the levels of pro-SFTPB. n = 3–5 mice per group. The level of pro-SFTPB was increased, peaking on day 10, across all source materials. (F) Uniform manifold approximation and projection (UMAP) embedding of single-cell transcriptomes from 77,656 cells from 5 control mice (on day 0) and 20 bleomycin-induced mouse lungs (on days 3, 7, 14, 28, n = 5 mice per group) annotated by cell type. (G and H) Density and dot plots of Sftpb mRNA expression levels. (I) Changes in the expression of Sftpb mRNA in alveolar type 2 epithelial cells (AT2) by pseudo-bulk analysis. n = 5 mice per group. (J) Dendrogram of high dimensional weighted correlation network analysis in AT2 cells of mice on days 0, 3, and 7. (K and L) The 10 most significantly (P < 0.05) enriched terms in GO biological process and KEGG pathways in 126 genes covarying with Sftpb. (B–E and I) The boxes indicate interquartile ranges (75% and 25%) and medians; the upper and lower whiskers represent the 10% and 90% points, respectively. The expression levels were compared by ANOVA, and Dunnett’s method was applied to adjust for the ANOVA P values. *P < 0.05; **P < 0.01.