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

Abstract

Background 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.

1. Introduction

Ankylosing spondylitis (AS) is an autoimmune disease primarily affecting the axial skeleton and adjacent soft tissues including tendons and ligaments. AS presents with a range of clinical features, the most common of which are chronic back pain and progressive spinal stiffness. In the advanced stages, patients may experience severe symptoms such as impaired spinal mobility and postural deformities, which significantly impact their quality of life. Current treatments for AS fail to reverse structural damage to the spine and other affected joints. Thus, more effective therapeutic strategies are needed to suppress systemic inflammation throughout all stages of disease progression, promote spinal repair, and reduce functional limitations.

Extensive studies have demonstrated the potential of mesenchymal stem cells (MSCs) in immune modulation and tissue repair, highlighting their promise as a therapeutic option for AS. Single-cell sequencing (scRNA-seq) has been utilized to compare MSCs derived from bone marrow, adipose tissue, umbilical cord, and skin, revealing that umbilical cord-derived MSCs exhibit superior immunosuppressive properties [1, 2], thereby making them a promising source for treating autoimmune diseases. Clinical studies have already reported the safety and efficacy of human umbilical cord MSCs (hUC-MSC) in AS patients, although the precise mechanisms underlying their therapeutic effects remain unclear.

Recent scRNA-seq studies on hUC-MSCs have uncovered multiple subpopulations with distinct phenotypes, gene expression profiles, and functional properties. In-depth analyses of these differences enhance our understanding of hUC-MSCs heterogeneity and provide insights into the mechanisms underlying their clinical applications [3]. Zhang et al. identified two distinct subpopulations of Wharton’s jelly cells. One subpopulation exhibited enriched gene expression associated with immunomodulation, cell proliferation, and differentiation, while the other was enriched in genes related to extracellular matrix.