Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapeutic efficacy for rheumatoid arthritis

Mesenchymal stem/stromal cell (MSC) therapy holds promise as a therapeutic strategy for rheumatoid arthritis (RA). However, the loss of secretory function following cell delivery has significantly restricted its clinical application. Our preliminary studies confirmed that endoplasmic reticulum stress (ERS)-MSCs greatly inhibited RA follicular helper T cells (Tfh) through cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) pathway activation via an unknown molecular mechanism, demonstrating the therapeutic effects of ERS-modified MSCs on RA. To compare their therapeutic efficacy, thapsigargin (TG)-stimulated or unstimulated MSCs were transplanted into collagen-induced arthritis (CIA) mice. Joint inflammation was evaluated from both general and histological aspects. Splenocytes were isolated, and flow cytometry was performed to assess the proportions of T helper 1 (Th1), Th17, and Tfh subsets. Additionally, the levels of TNF-α in mouse serum were measured using ELISA. For mechanistic exploration, the TRRUST and Cistrome Data Browser databases were used to analyse transcription factors related to COX2 regulation, as well as target genes regulated by activating transcription factor 4 (ATF4). To identify the most effective treatment concentration and duration for inducing ERS, we conducted a concentration and time gradient analysis for TG treatment via qRT‒PCR and a CCK‒8 assay. Then, western blotting and qRT‒PCR were employed to determine the level of ATF4 in ERS-MSCs. To verify the function of ATF4 in vivo, ATF4-overexpressing MSCs were transplanted into CIA mice, the levels of joint inflammation as well as the proportions of Th1, Th17 and Tfh subsets were analysed. To clarify the molecular regulatory mechanism leading to ATF4 activation, the protein levels of protein kinase RNAs, such as endoplasmic reticulum kinase (PERK)/phosphorylated-PERK (p-PERK) and eukaryotic initiation factor 2α (eIF2α)/phosphorylated-eIF2α (p-eIF2α) were examined. Furthermore, the levels of ATF4 and eIF2α/p-eIF2α were assessed after PERK blockade. Mitochondrial stress was subsequently examined in ERS-MSCs. Finally, when blocking ERS and mitochondrial stress were inhibited separately or simultaneously, the levels of ATF4 and eIF2α/p-eIF2α were reevaluated. Compared with MSCs, ERS-MSCs exhibited greater therapeutic efficacy in CIA mice. Public databases and bioinformatics analyses confirmed the regulatory role of ATF4 in COX2, and experimental methods further demonstrated that ATF4-transfected MSCs alleviated joint inflammation in CIA mice. We also demonstrated that during ERS induction, PERK-mediated eIF2α phosphorylation contributes to the activation of ATF4. Furthermore, mitochondrial stress was also provoked in ERS-MSCs, and ERS synergistically regulated ATF4. Compared with unmodified MSCs, ERS-MSCs exhibited enhanced immunosuppressive potency, primarily through COX2 overexpression, which was regulated by ATF4 activation. Moreover, ERS and mitochondrial stress jointly regulated ATF4 expression. This study reveals a novel role of ATF4 in enhancing the secretory properties of MSCs and has thereby presents a promising MSC-based therapeutic strategy for the treatment of RA.