# Prolonged TNF-α stimulation induces a PD-1–associated exhaustion-like phenotype in mesenchymal stromal cells

## TL;DR

Prolonged exposure to TNF-α causes mesenchymal stromal cells to adopt an exhausted-like state, impairing their function in chronic inflammation.

## Contribution

This study reveals that chronic TNF-α stimulation leads to a PD-1-associated exhaustion-like dysfunction in mesenchymal stromal cells.

## Key findings

- Prolonged TNF-α stimulation increases Pd-1 and Ctla-4 expression, indicating an exhaustion-like state in MSCs.
- Chronic stimulation suppresses immunoregulatory genes and metabolic pathways while activating NF-κB and Stat3.
- PD-1 upregulation is linked to MSC dysfunction under chronic inflammatory conditions.

## Abstract

Mesenchymal stromal cells (MSCs) have emerged as promising therapeutic agents for inflammatory diseases because of their potent immunomodulatory properties. Although acute inflammation transiently enhances MSC functionality, the impact of chronic inflammatory exposure remains poorly defined. In this study, we investigated the effects of sustained TNF-α stimulation and indirect co-culture with M1 macrophages on MSC behavior. Comprehensive gene expression profiling was performed to assess the changes in immunoregulatory, apoptotic, and metabolic pathways. To determine functional reversibility, we also evaluated MSCs following the withdrawal of TNF-α. Short-term exposure led to upregulation of Tgf-β, Il-10, and Fasl, whereas prolonged stimulation suppressed these genes and significantly increased the expression of immune checkpoint genes Pd-1 and Ctla-4, indicative of an exhaustion-like phenotype. This phenotypic shift was associated with sustained NF-κB activation, upregulation of Stat3 and Ap-1, suppression of mTORC1/2 components, decreased Pd-l1 expression, and increased Pd-1 expression, raising the possibility that PD-1 upregulation is associated with MSC dysfunction under chronic inflammatory stress. These findings revealed that prolonged stimulation (48 h) induces an exhaustion-like dysfunction state in MSCs, characterized by checkpoint activation, transcriptional repression, and metabolic dysfunction. PD-1 may serve as a biomarker associated with inflammation-induced MSC impairment.