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Backgroud <p>This study systematically evaluated the immunomodulatory function of PD-L1-positive mesenchymal stem cells (PD-L1(+) MSCs) using single-cell RNA sequencing (scRNA-seq) and investigated their roles in suppressing inflammation and regulating pathological bone formation in curdlan-induced SKG ankylosing spondylitis (AS) mouse models.</p> Methods <p>scRNA-seq identified MSC subpopulations with high immunomodulatory capacity and key biomarker PD-L1 for subpopulation classification. In vitro co-culture experiments were conducted to evaluate the effects of MSC subpopulations on T-cell proliferation and TNF-α levels. In vivo experiments were performed in forty-eight SKG mouse models to analyze the effects of MSC subpopulations on joint inflammation scores, T-cell subset proportions, inflammatory cytokines, histopathology, and pathological bone formation.</p> Results <p>scRNA-seq revealed significant heterogeneity in MSCs under inflammatory stimulation, with the immunomodulatory subpopulation exhibiting high expression of PD-L1 and IDO. In vitro experiments demonstrated that PD-L1(+) MSCs significantly suppressed T-cell proliferation and reduced TNF-α levels. Joint redness and swelling scores showed that the PD-L1(+) MSC group exhibited the most significant improvement in arthritis, while the IL-17Ai, PD-L1(-) MSC, and MSC groups also effectively reduced inflammation, with significantly lower scores than the model control(MC) group. Histological analysis revealed severe inflammatory cell infiltration in the MC group, while the IL-17Ai, PD-L1(+) MSC, and MSC groups exhibited reduced infiltration. Immunohistochemical analysis further confirmed these findings, with PD-L1(+) MSCs exhibiting a significant reduction in TNF-α and IL-17A-positive cells (P &lt; 0.0001 and P &lt; 0.01, respectively).PD-L1(+) MSCs regulated immune responses by reducing Th17 cell proportions, increasing Th2 and Treg cell proportions, and significantly lowering pro-inflammatory cytokines IFN-γ, IL-17A, and TNF-α. MicroCT analysis indicated that the PD-L1(+) MSC, MSC, and IL-17Ai group effectively suppressed pathological bone formation through immunomodulation, whereas the PD-L1(-) MSC group showed weaker effects, underscoring the importance of PD-L1 in regulating bone formation.</p> Conclusion <p>hUC-MSCs demonstrated significant therapeutic effects in the AS mouse model, particularly the PD-L1(+) MSCs, which inhibited joint inflammation and pathological new bone formation through immunomodulatory mechanisms. These findings provide valuable insights into the therapeutic mechanisms of AS treatment.</p>

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Single—cell RNA sequencing identifies PD—L1 + mesenchymal stem cells with enhanced immunomodulatory capacity and alleviated the degree of ectopic new bone formation in ankylosing spondylitis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月29日

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  • Xiqing Luo,
  • Liuzhong Zhou,
  • Xianghui Wen,
  • Jinwei Li,
  • Dong Liu,
  • Budian Liu,
  • Shenghui Wen,
  • Jieruo Gu

摘要

Backgroud

This study systematically evaluated the immunomodulatory function of PD-L1-positive mesenchymal stem cells (PD-L1(+) MSCs) using single-cell RNA sequencing (scRNA-seq) and investigated their roles in suppressing inflammation and regulating pathological bone formation in curdlan-induced SKG ankylosing spondylitis (AS) mouse models.

Methods

scRNA-seq identified MSC subpopulations with high immunomodulatory capacity and key biomarker PD-L1 for subpopulation classification. In vitro co-culture experiments were conducted to evaluate the effects of MSC subpopulations on T-cell proliferation and TNF-α levels. In vivo experiments were performed in forty-eight SKG mouse models to analyze the effects of MSC subpopulations on joint inflammation scores, T-cell subset proportions, inflammatory cytokines, histopathology, and pathological bone formation.

Results

scRNA-seq revealed significant heterogeneity in MSCs under inflammatory stimulation, with the immunomodulatory subpopulation exhibiting high expression of PD-L1 and IDO. In vitro experiments demonstrated that PD-L1(+) MSCs significantly suppressed T-cell proliferation and reduced TNF-α levels. Joint redness and swelling scores showed that the PD-L1(+) MSC group exhibited the most significant improvement in arthritis, while the IL-17Ai, PD-L1(-) MSC, and MSC groups also effectively reduced inflammation, with significantly lower scores than the model control(MC) group. Histological analysis revealed severe inflammatory cell infiltration in the MC group, while the IL-17Ai, PD-L1(+) MSC, and MSC groups exhibited reduced infiltration. Immunohistochemical analysis further confirmed these findings, with PD-L1(+) MSCs exhibiting a significant reduction in TNF-α and IL-17A-positive cells (P < 0.0001 and P < 0.01, respectively).PD-L1(+) MSCs regulated immune responses by reducing Th17 cell proportions, increasing Th2 and Treg cell proportions, and significantly lowering pro-inflammatory cytokines IFN-γ, IL-17A, and TNF-α. MicroCT analysis indicated that the PD-L1(+) MSC, MSC, and IL-17Ai group effectively suppressed pathological bone formation through immunomodulation, whereas the PD-L1(-) MSC group showed weaker effects, underscoring the importance of PD-L1 in regulating bone formation.

Conclusion

hUC-MSCs demonstrated significant therapeutic effects in the AS mouse model, particularly the PD-L1(+) MSCs, which inhibited joint inflammation and pathological new bone formation through immunomodulatory mechanisms. These findings provide valuable insights into the therapeutic mechanisms of AS treatment.