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. 2024 Sep 2:15:1440918.
doi: 10.3389/fimmu.2024.1440918. eCollection 2024.

Tff1-expressing Tregs in lung prevent exacerbation of Bleomycin-induced pulmonary fibrosis

Masaaki Okamoto  1   2 , Ayumi Kuratani  1   2 , Daisuke Okuzaki  3 , Naganori Kamiyama  4 , Takashi Kobayashi  4   5 , Miwa Sasai  1   2   6 , Masahiro Yamamoto  1   2   6
Affiliations

Tff1-expressing Tregs in lung prevent exacerbation of Bleomycin-induced pulmonary fibrosis

Masaaki Okamoto et al. Front Immunol. .

Abstract

Bleomycin (BLM) induces lung injury, leading to inflammation and pulmonary fibrosis. Regulatory T cells (Tregs) maintain self-tolerance and control host immune responses. However, little is known about their involvement in the pathology of pulmonary fibrosis. Here we show that a unique Treg subset expressing trefoil factor family 1 (Tff1) emerges in the BLM-injured lung. These Tff1-expressing Tregs (Tff1-Tregs) were induced by IL-33. Moreover, although Tff1 ablation in Tregs did not change the pathological condition, selective ablation of Tff1-Tregs using an intersectional genetic method promoted pro-inflammatory features of macrophages in the injured lung and exacerbated the fibrosis. Taken together, our study revealed the presence of a unique Treg subset expressing Tff1 in BLM-injured lungs and their critical role in the injured lung to ameliorate fibrosis.

Keywords: Bleomycin; Tff1; Treg; VeDTR; fibrosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Lung Tregs express Tff1 in BLM-administered lung. (A–F) CD45+ cells from lungs of naïve and Bleomycin-administered WT mice were applied to scRNA seq analysis. Data shows pooled samples (n = 3 biologically independent mice). See also Supplementary Figure 1 (A) UMAP plots of lung T cells indicating 6 clusters: whole (left), naïve group (center) and BLM group (right). (B) UMAP plots indicating Foxp3 expression. (C) Heatmap indicating feature genes of 6 clusters. (D) Volcano plots showing upregulated DEGs (FC > 1.5, q-value < 0.05) in the Treg cluster versus other clusters in the BLM group (left) and the Treg cluster in the BLM group versus the naïve group (right). DEGs lists are described in Supplementary Tables 1 , 2 . (E) Venn diagram of upregulated genes of indicated samples. For Bulk RNA-seq analysis, GFP+ CD4 T cells (Tregs) from spleens and lungs of Bleomycin-administered FDG mice were isolated. Data shows pooled samples (n = 3 biologically independent mice). Transcriptome profiles of Bulk RNA-seq are described in Supplementary Table 3 and upregulated DEGs of lung Tregs were identified by FC > 20. (F) UMAP plots indicating Tff1 expression. (G) GFP-CD4 T cells (Tconv) and GFP+CD4 T cells (Tregs) were isolated from spleens and lungs of naïve and BLM-administered FDG mice. Tff1 expression was measured by Q-PCR. (H) Tregs (Lin (B220, CD8a, CD11b, CD11c, NK1.1)-DAPI-GFP+) were isolated from lungs of naïve and BLM-administered FDG mice, fixed and permeabilized, and stained with DAPI and α-Tff1 antibody.
Figure 2
Figure 2
Generation of Foxp3-Cre/Tff1-Flp/VeDTR mice. (A) The genome editing strategy of Foxp3 and Tff1-dependent intersectional expression of YFP and DTR. (B–F) Foxp3-Cre/Tff1-Flp/VeDTR mice were i.t. administered with BLM. (B, C) Frequency of YFP+ in lung CD4+ T cells was measured over time. (B) Representative plot of day 0 (naïve) and day 14 post BLM administration. (C) Total results in indicated time points (n = 6 each). (D) Foxp3-Cre/Tff1-Flp/VeDTR mice were i.t. administered with PBS or BLM (n = 3 each). Frequency of YFP+ in CD4+ T cells of indicated tissues was determined on d21. (E) Frequency of YFP+ in lung CD4+ T cells was measured at indicated time points (n = 6 each). (F) Saline or FTY720 (4 µg) or daily i.p. administered in BLM-treated mice from day 0 (n=5 each). Frequency of YFP+ in CD4+ T cells of indicated tissues was determined on d14 Data means with SD. significance assessment: unpaired two-tailed Student’s t-test. ***; p<0.001.
Figure 3
Figure 3
IL-33 is involved in Tff1 expression in lung Tregs in vitro and in vivo. (A) CD25 and GFP expression in CD4+ T cells from spleen (Sp) and lung (Lu) of naïve FDG mice were measured. Representative plot of two independent experiments. (B) ST2 expression in CD25+CD4+ T cells from spleen and lung of naïve Foxp3-Cre/Tff1-Flp/VeDTR mice was measured. Representative plot of two independent experiments. (C) YFP-CD25+CD4+ T cells from lung of naïve Foxp3-Cre/Tff1-Flp/VeDTR mice (n = 3) were cultured in the presence of indicated cytokines and α-CD3/CD28 antibody-bounded Dynabeads. 6 days later, YFP expression was measured. Representative plot (spleen: top left, lung: bottom left), and total result (spleen: top right, lung: bottom right). (D, E) Foxp3-Cre/Tff1-Flp/VeDTR mice were i.n. administered PBS (n = 4) or IL-33 (n = 6). (D) Representative image of HE staining of lung section. (E) Frequency of YFP+ in lung CD4+ T cells were measured Data of (C, E) are means with SD. significance assessment: (C) One-way ANOVA with a post-Tukey’s test and (E) unpaired two-tailed Student’s t test. **; p<0.01, *; p<0.05 and ns; not significant.
Figure 4
Figure 4
Ablation of Tff1-Tregs progress fibrosis. (A–D) Foxp3-Cre/Tbff1-Flp/VeDTR mice were administered or not administered BLM. PBS or DT (100ng) was i.p. administered every 3 days from day 7. Each analysis was conducted on day 21. (A) The frequency of YFP+ cells in lung CD4+ T cells was measured by flow cytometry. Representative plot (left) and total results (right) (PBS: n = 9, DT: n = 10). (B) Hydroxyproline levels in lung were measured. (BLM(-) PBS: n = 7 BLM(-) DT: n = 7, BLM(+) PBS: n = 11, BLM(+) DT: n = 13). (C) HE and Azan staining of lung section of BLM-non-administered mice and (D) BLM-administered mice. Data of (A, B) are means with SD. significance assessment: unpaired two-tailed Student’s t test. ***; p<0.001, *; p<0.05 and ns; not significant.
Figure 5
Figure 5
Ablation of Tff1-Tregs changes immune landscape. (A–D) Foxp3-Cre/Tbff1-Flp/VeDTR mice were administered BLM. PBS or DT (100ng) was i.p. administered every 3 days from day 7 (n = 3 each). Lung CD45+ cells were subjected to CyTOF analysis on day 21. UMAP-plot (A) of whole condition and (B) of PBS (left) and DT condition (right). Expression of indicated genes in (C) population “a” and CD4+ T-1, (D) “b” (E) “c” and (F) “d”, which are annotated in (B). See also Supplementary Figure 3 .
Figure 6
Figure 6
Ablation of Tff1-Tregs enhance proinflammatory features of myeloid cells. (A–C) Foxp3-Cre/Tbff1-Flp/VeDTR mice were administered BLM. PBS (n = 7) or DT (100ng) (n = 6)was i.p. administered every 3 days from day 7. Lung CD45+ cells were subjected to flow cytometry analysis on day 21. (A) Gating strategy for discriminating the indicated cell populations. (B) Cell number of each population. (C) Expression level of the CD80 and CD86 in indicated population. Data of (B, C) are means with SD. Statistical significance assessment: unpaired two-tailed Student’s t test. **; p<0.01, *; p<0.05 and ns; not significant.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by Japan Science and Technology Agency (JPMJFR206D and JPMJMS2025); Agency for Medical Research and Development (JP20fk0108137, JP23fk0108682, and JP223fa627002); Ministry of Education, Culture, Sports, Science and Technology (24K10257); the program from Joint Usage and Joint Research Programs of the Institute of Advanced Medical Sciences, Tokushima University; Takeda Science Foundation; Mochida Memorial Foundation; Astellas Foundation for Research on Metabolic Disorders; Naito Foundation; the Chemo-Sero-Therapeutic Research Institute; Research Foundation for Microbial Diseases of Osaka University; BIKEN Taniguchi Scholarship; The Nippon Foundation - Osaka University Project for Infectious Disease Prevention; Joint Research Program of Research Center for Global and Local Infectious Diseases of Oita University (2021B06); the Research Fellow of Scholarship for Doctoral Students in Immunology.

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