Role of STING in mesenchymal stromal cell senescence

Abstract

Mesenchymal stromal cell (MSC) is a promising biotherapeutic agent with strong potency in self-renewal, differentiation, and immunoregulation, but high quality of MSCs is required for satisfying outcomes. MSC senescence frequently occurs in the inflammatory environment or under oxidative stress, posing threats to the quality and therapeutic efficacy of MSCs. Senescence-associated secretory phenotype (SASP), manifesting as increased secretion of inflammatory cytokines in senescent MSCs, impairs their immunosuppressive function. To date, the mechanism of MSC senescence is far from fully elucidated. STING activation was recently found as an important pathway that promotes cellular senescence. However, its role in mediating MSC senescence has not been reported. Thus, it is worthwhile to explore the effect of STING activation on MSC senescence, the intervention of which could potentially represent a new strategy for maintaining MSC stemness.

Herein, this study explored the effect of STING activation on MSC senescence. Human bone marrow-derived MSCs were stimulated with poly(dA:dT), a synthetic double-stranded DNA, and a specific STING ligand 2’3’-cGAMP (cGAMP). STING activation via poly(dA:dT) impaired MSC proliferation and increased p21 expression. The proportion of cells at the S phase was reduced, indicating the occurrence of cell cycle arrest. STING activation increased senescence biomarker SA-β-gal activity in MSCs and promoted the secretion of inflammatory cytokines, suggesting that STING activation induced MSC senescence. The reduced cell proliferation and cytokine secretion could be recapitulated by cGAMP. Blocking STING signaling via specific inhibitor H151 or STING knockdown attenuated STING-induced senescence phenotype. STING activation in MSCs also compromised their immunoregulatory function in suppressing T cell proliferation.

STING activation could trigger autophagy, a process involved in MSC senescence. Thus, the effect of autophagy inhibition on STING activation and MSC senescence was further explored. STING signaling and production of inflammation cytokines were enhanced in human bone marrow-derived MSCs treated with autophagy inhibitors. Besides, autophagy inhibition augmented mitochondrial superoxide production in STING-activation MSCs, suggesting autophagy played a role in regulating superoxide production in MSCs and attenuating STING signaling in its downstream, thus protecting the cells from senescence.

To explore the intrinsic effect of STING activation on MSC senescence, MSCs were isolated from the bone marrow of wild type (WT), STING gain-of-function (carrying heterozygous N153S mutation), and STING knockout mice. Bone marrow MSCs from mice carrying the N153S mutation showed spontaneous STING activation and exhibited impaired proliferation under normoxic culture conditions compared with WT and STING knockout mice. SA-β-gal staining further demonstrated that STING knockout MSCs were more resistant to senescence in atmospheric oxygen condition, suggesting a central role of STING in promoting MSC senescence.

In conclusion, our study proved the role of STING activation in promoting MSC senescence and compromising their immunoregulatory function, providing a new target for monitoring and preventing MSC senescence, which may contribute to improving the performance of MSCs in future clinical applications.