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 5

Increased levels of immature SFTPB protein in patients with PPF.

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Increased levels of immature SFTPB protein in patients with PPF. (A) A r...
(A) A representative image of immunohistochemistry for SFTPB using lung sections from controls and PPF cases. In controls, SFTPB-positive alveolar epithelial cells were scattered over the alveolar surface, while SFTPB-positive alveolar epithelial cells with reactive atypia covered the alveolar surface in fibrotic areas. (B–E) Western blotting for evaluating SFTPB in 10 lung tissue specimens, including 5 PPF surgical specimens and 5 control tissues (surgical specimens from patients with lung cancer). (F–H) Western blotting for evaluating SFTPB in the serum of HCs (n = 5) and PPF cases (n = 6). (I and J) Western blotting for evaluating SFTPB in serum EVs from HCs (n = 5) and PPF cases (n = 6). The same samples were used as in the study of serum. (K and L) Western blotting for evaluating SFTPB between the serum and serum EVs in PPF cases (n = 6). The same samples were used as in the studies of serum and serum EVs. Serum samples per lane were generated from 0.01 μL of serum, while serum EV samples per lane were generated from 70 μL of serum. (B–L) Patient characteristics are shown in Supplemental Tables 9 and 10. The intensity of the SFTPB band was evaluated using ImageJ (NIH) and normalized to levels of β-actin in lung tissue specimens. Differences between 2 groups were compared using 2-tailed Student’s t test. *P < 0.05; **P < 0.01.