A distinct “repair” role of regulatory T cells in fracture healing

doi:10.1007/s11684-023-1024-8

Front. Med.

2024, Vol. 18

Issue (3) : 516-537 https://doi.org/10.1007/s11684-023-1024-8

A distinct “repair” role of regulatory T cells in fracture healing

Tingting Wu^1,², Lulu Wang^1,², Chen Jian^1,², Zhenhe Zhang³, Ruiyin Zeng³, Bobin Mi³, Guohui Liu³, Yu Zhang^1,², Chen Shi^1,²(

)

¹. Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
². Hubei Province Clinical Research Center for Precision Medicine for Critical Illness, Wuhan 430022, China
³. Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

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Abstract

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Keywords regulatory T cells fracture healing amphiregulin non-union osteogenesis

Corresponding Author(s): Chen Shi

Just Accepted Date: 28 November 2023 Online First Date: 15 March 2024 Issue Date: 17 June 2024

Cite this article:

Tingting Wu,Lulu Wang,Chen Jian, et al. A distinct “repair” role of regulatory T cells in fracture healing[J]. Front. Med., 2024, 18(3): 516-537.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-023-1024-8
https://academic.hep.com.cn/fmd/EN/Y2024/V18/I3/516

Fig.1 Treg accumulation in fractured bone. (A) Flow scatter diagram of CD4⁺ T cells in the spleen, peripheral blood, and bone callus before fracture (day 0) and days 1, 3, 5, 14, and 21 after fracture. Numbers indicate the proportion of cells in the frame. (B–D) Quantitative analysis of CD4⁺ T cells in the spleen (B), peripheral blood (C), and bone callus (D) at different time points after fracture (n = 3). (E) Flow scatter diagram of Tregs in the spleen, peripheral blood, and bone callus before fracture (day 0) and on days 1, 3, 5, 14, and day 21 after. Numbers indicate the proportion of cells in the frame. (F–H) Proportions of Tregs in the spleen (F), peripheral blood (G), and bone callus (H) at different time points after fracture. Five mice were set as one group for all tissues. Data are shown as means ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig.2 Sources of Tregs in fractured bone. (A, B) Percentages of CD4⁺ T cells (A) and Tregs (B) in the bone callus (n = 4). FTY720 or solvents were administrated on day 1 before fracture and days 1, 2, 3, and 4 after fracture. Bone callus was collected on day 5 after fracture with the extraction of lymphocytes. (C) Histograms depict the expression of neuropilin-1 and Helios in Tregs from the bone callus and spleen on day 5 post fracture. (D) FCM graphs of Ki67 expression in spleen and bone callus Tregs and Tconvs. Bone callus and spleen were dissected for the analysis 5 days post fracture. Numbers indicate the proportion of cells in the frame. (E) Quantitative analysis of Ki67 in the spleen and bone callus Treg and Tconv cell subsets (n = 3). (F) FCM graphs of EdU expression in spleen and bone callus Tregs and Tconvs. Numbers indicate the proportion of cells in the frame. A total of 1 mg EdU was intraperitoneally injected to the mice, and FCM analysis was performed on 24 h post EdU administration. (G) Quantitative analysis of EdU in spleen and bone callus Tregs and Tconvs (n = 3). Data are shown as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig.3 Tregs from bone callus promoted BMSC proliferation and osteogenesis. (A) Proliferation of BMSCs cultured with different sources of Tregs was tested via EdU assay. The higher the proportion of red dots, the more proliferative the cells. Scale bar = 200 µm. (B) ALP staining of BMSCs in different treatments after culturing with osteogenic induction medium for 14 days. Scale bar = 200 µm. (C) Quantification analysis of the percentage of EdU⁺ cells (n = 5). (D) Quantitative analysis of ALP-positive area calculated using ImageJ software (n = 5). Data are shown as means ± SD. *, P < 0.05.

Fig.4 Depletion of Tregs compromised fracture healing. (A) X-Ray image of fractured mice. On days 1, 3, 5, 7, 14, and 21, the mice were anesthetized using 0.3% pentobarbital (w/v) and scanned via X-ray. (B, C) Micro-CT three-dimensional reconstructed images of the fractured femur on day 21 post fracture. The mice were sacrificed, and the femur was separated for micro-CT scanning. Four mice were included in each group. (D) Calculated BMD, TV, BV, and BV/TV for each group (n = 5). The ROIs were selected as areas 1 mm above and below the fracture gap. (E) mRNA levels of osteogenesis-related proteins (collagen I, OCN, and RUNX2) in bone callus (n = 5). Mice were sacrificed on day 21 post fracture, and the bone callus was analyzed via qRT-PCT. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig.5 Bone callus Tregs displayed distinct TCR repertoire and transcriptome. (A) Left, t-SNE projection of spleen CD4⁺ T cells displaying the main cell clusters. The purple dots denote the Treg clusters. Each dot represents a single cell, and each color indicates a different cluster. CD4⁺ T cells were sorted via FCM on day 5 post fracture, with the cell number reaching 10591. Right, t-SNE projection of bone callus CD4⁺ T cells and Foxp3⁺ Tregs, with the cell number reaching 9916. (B) Identical paired CDR3 α and CDR3 β sequences in bone callus Tregs. (C) GSEA revealing the Gene Ontology enrichment of the biological process category in bone callus Tregs vs spleen Tregs. The top 20 pathways are listed and ranked by normalized enrichment score. (D) Heat map of selected differential genes between bone callus and spleen Tregs. Averaged from three independent experiments. (E) Cytofluorometric analyses of amphiregulin in bone callus and spleen Tregs (n = 4). Data are shown as means ± SD. *, P < 0.05. Areg indicated as amphiregulin in the graphs.

Fig.6 Amphiregulin promoted proliferation and osteogenesis of BMSCs. (A) After the treatment with different concentrations of amphiregulin from 0 ng/mL to 2.5 ng/mL, EdU assay was performed in 48 h. Scale bar = 200 µm. (B) Quantification analysis of the percentage of EdU-positive cells (n = 5). (C) ALP staining of BMSCs treated with different concentrations of amphiregulin from 0 ng/mL to 100 ng/mL after culturing with osteogenic induction medium for two weeks. (D) Alizarin Red staining of BMSCs treated with different concentrations of Areg from 0 ng/mL to 100 ng/mL after culturing with osteogenic induction medium for six weeks. (E) Quantitative analysis of ALP-positive area calculated using ImageJ software (n = 5). (F) Quantitative analysis of Alizarin Red-positive area calculated using ImageJ software (n = 5). (G) Protein levels of AKT and p-AKT in BMSCs treated with amphiregulin with or without the PI3K inhibitor LY294002 and AKT inhibitor deguelin at different time points (0, 30, 60, and 90 min). (H) Protein expressions of osteogenic markers RUNX2 and BMP-2 (0, 3, and 7 days). Data are shown as means ± SD. *, P < 0.05; **, P < 0.01. Areg indicated as amphiregulin in the graphs.

Fig.7 Amphiregulin reversed the compromised healing caused by Treg depletion. (A) Mouse model and administration protocol. (B) X-Rays images of fracture healing process among different groups on days 7, 14, and 21 post fracture. The mice were anesthetized via injection of sodium pentobarbital to capture X-ray images. (C) Micro-CT three-dimensional reconstructed images of fractured femur on day 21 post fracture. The mice were sacrificed, and the femur was separated for micro-CT scanning. Four mice were included in each group. (D) Calculated BMD, TV, BV, and BV/TV in each group (n = 5). The ROIs were selected as areas 1 mm above and below the fracture gap. (E–G) Serum levels of OCN (E), ALP (F), and B-ALP (G) in different groups (n = 3). (H) Representative histological evaluation of callus formation on day 21. The calluses were stained with H&E/TRAP/AB. Scale bar for HE and TRAP = 200 µm; scale bar for AB = 100 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Areg indicated as amphiregulin in the graphs.

Fig.8 Exploration of amphiregulin for human fracture healing. (A) Flow scatter diagrams of human CD4⁺ T (hCD4⁺ T) cells in peripheral blood and bone callus of fracture patients. Numbers indicate the proportion of cells in the frame. (B) Proportions of hCD4⁺ T cells in peripheral blood and bone callus of fracture patients (n = 15). (C) Flow scatter diagram of Tregs (hTregs) in peripheral blood and bone callus of fracture patients. Numbers indicate the proportion of cells in the frame. (D) Proportions of hTregs of fracture patients (n = 15). (E) mRNA levels of amphiregulin in blood and bone callus Tregs (n = 8). (F) EdU assay of hUC-MSCs treated with different concentrations of amphiregulin for 48 h. Scale bar = 200 µm. (G) Quantification analysis of the percentage of EdU-positive cells (n = 5). (H) ALP and Alizarin Red staining of hUC-MSCs treated with different concentrations of amphiregulin. Scale bar = 100 µm. (I,J) Quantitative analysis of ALP and Alizarin Red-positive area calculated using ImageJ software (n = 5). Data are shown as means ± SD. *, P < 0.05; ***, P < 0.001. Areg indicated as amphiregulin in the graphs.

Fig.9 Proposed mechanism of Treg-mediated fracture healing process. PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; BGLAP, osteocalcin.

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[1]	Xiaoyu Wang,Yuxuan Gao,Haigang Shi,Na Liu,Wei Zhang,Hongbo Li. Influence of the intensity and loading time of direct current electric field on the directional migration of rat bone marrow mesenchymal stem cells[J]. Front. Med., 2016, 10(3): 286-296.

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