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

Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapy for rheumatoid arthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapy for rheumatoid arthritis Jiaqing Liu, Xing Zhang, Xiangge Zhao, Jinyi Ren, Huina Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5620379/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 May, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background Mesenchymal stem/stromal cell (MSCs) therapy represents a potential therapeutic tool to treat RA, but loss of secretory property post delivery restricted clinical application. It has been verified that endoplasmic reticulum stress (ERS)-MSCs exhibited better inhibition on rheumatoid arthritis (RA) T follicular helper cells (Tfh) via cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) activation with unknown molecular mechanism, particulary the overall outcome of ERS-modified MSCs on RA. Methods To compare the therapeutic efficacy, thapsigargin (TG)-stimulated or unstimulated MSCs were transplantated into collagen-induced arthritis (CIA) mice. Joint inflammation was evaluated from general and histological aspects. Splenocytes were isolated and flow cytometry was performed to assess the proportion of T helper 1 (Th1), Th17 and Tfh subsets. During mechanism exploration, TRRUST and Cistrome Data Browser databases were used to analyze transcription factors related to COX2 regulation, as well as target genes regulated by activating transcription factor 4 (ATF4). Then western blot and qRT-PCR were employed to determine the level of ATF4 in ERS-MSCs. To verify the function of ATF4 in vivo , ATF4-overexpression MSCs were transplanted to CIA mice, joint inflammation, Th1, Th17 and Tfh subsets were analysed. To clear the molecular regulatory mechanism leading to ATF4 activation, protein levels of protein kinase RNA like endoplasmic reticulum kinase (PERK)/phosphorylated-PERK (p-PERK) and eukaryotic initiation factor 2α (eIF2α)/phosphorylated-eIF2α (p-eIF2α) were examined. Besides, ATF4 and eIF2α/p-eIF2α were checked after PERK blocking. Subsequently, mitochondrial stress was checked in ERS-MSCs. At last, blocking ERS and mitochondrial stress separately or simultaneously, ATF4 and eIF2α/p-eIF2α were checked again. Results Compared with MSCs, ERS-MSCs exhibited better therapeutic efficacy in CIA mice. Public databases and bioinformatics analysis confirmed the regulatory role of ATF4 on COX2 and experimental methods further confirmed ATF4-transfected MSCs diminished the joint inflammation of CIA mice. We also demonstrated that during ERS induction, PERK-mediated eIF2α phosphorylation contributes to elevated ATF4 expression. Besides, mitochondrial stress was also provoked in ERS-MSCs, coupling with ERS synergistically regulated ATF4. Conclusions ERS-MSCs exhibited better immunosuppresive ability than un-pretreated MSCs through COX2 overexpression, which was regulated by ATF4. Besides, ERS and mitochondrial stress co-regulate ATF4 expression. This study established a new role of ATF4 in promoting secretory properties of MSC and provided a promising MSC-based therapeutic strategy for RA treatment. MSCs ATF4 rheumatoid arthritis reticulum-mitochondria coupling stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mesenchymal stromal/stem cells (MSCs) have been serving as a valuable source for treatment of immune-mediated disorders [ 1 , 2 ]. MSC-based therapies have encountered translational hurdles for the decreased immunosuppressive capacities post delivery [ 3 ]. Researchers are interested in developing new strategies to increase the immunosuppressive potential of MSCs in clinical applications [ 4 ]. Nowadays, many approaches have been proposed to enhance the therapeutic effects of MSCs [ 5 ]. Among them, pre-treated MSCs are easier to manipulate and have been studied in a variety of disease models [ 6 , 7 ]. Endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded or misfolded proteins in ER can lead to ER stress. ER stress functions as a double-edged sword, leading to apoptosis or resulting in cellular function changes, which determine cell function, fate and survival [ 8 ]. Disturbed ER homeostasis activates unfolded protein response (UPR) sensor proteins, including protein kinase RNA like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1 (IRE1α) and activating transcription factor 6 (ATF6) to restore the equilibrium. Activated PERK phosphorylates eukaryotic initiation factor 2α (eIF2α) which prevents translation of most mRNAs by inhibiting the initiation complex [ 9 , 10 ]. Concurrently, phosphorylation of eIF2α favors increased translation of selective mRNAs, and one of the increased protein-translations is activating transcription factor 4 (ATF4), a principal regulator which plays a crucial role in the adaptation to stresses through regulating the transcription of many genes [ 11 ]. Rheumatoid arthritis (RA) is a progressive and chronic autoimmune disease. Although the exact causes are unknown, the differentiation of CD4 + T lymphocytes into pathogenic CD4 + T subsets occupies a remarkable position in RA pathogenesis [ 12 , 13 ]. Some RA therapeutic medications have displayed ER stress-modulating properties. It has been verified that ER stress-modulating traditional Chinese medicines as potential new pharmaceutical drugs for RA clinical therapy [ 14 ]. Our previous results have shown that ER-stressed (ERS) MSCs displayed better immunomodulation effects in Tfh subsets from RA patients through elevated prostaglandin E2 (PGE2) secretion [ 13 ]. This study aimed at exploring the therapeutic effect of ERS-MSC on ameliorating the severity of arthritis in a mouse model of RA, together with the molecular mechanism of PGE2 higher expression, which could provide preclinical experimental data for the development of new stem cell drugs for the treatment of RA. Methods Isolation, culture, and identification of hUCMSCs Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) were isolated from healthy donors who gave birth and signed informed consents according to protocols reported previously [ 15 , 16 ]. The umbilical cord tissues were cut into small segments of about 1.0 mm3, then washed with PBS and centrifuged at 1900 r/min for 6 min. And cultured in an incubator at 37°C and 5% CO 2 concentration with DMEM/F12 (Meilunbio, China) consisting of 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA), 100 U/mL penicillin (Solarbio, China), and 100 mg/mL streptomycin (Solarbio, China). The primary cells were harvested when the confluence reached 80% and further characterized by flow cytometry (Agilent, USA) with following fluorescein-labeled antibodies: CD90-Perep, CD105-PE, CD45-FITC, CD34-APC, CD14-FITC, CD11b-APC, and HLA-DR-PE (eBioscience, USA). Isotype-matched control antibodies were used as methodology controls. After identification (Fig. S1 ), hUCMSCs were treated with TG for ERS induction and followed with verificaion of an ERS induction (data not shown), as mentioned previously [ 13 ]. Cells reaching the fifth passage were employed in the following experiments. Animals, disease induction and treatment Male adult DBA/1 mice at 6–8 weeks were purchased from Shanghai SLAC Laboratory Animals Company and housed under pathogen-free conditions at the Laboratory Animal Center of Dalian Medical University. Following a one-week acclimation period, mice undergoing the disease induction protocol were administered intradermal tail injections of a prepared collagen/Complete Freund adjuvant (CFA, Chondrex, USA) emulsion containing 1 mg/ml bovine type II collagen (Chondrex, USA) and 2 mg/ml at experimental day 0. Mice received a subsequent intradermal tail booster injection on day 21 with emulsion containing 1 mg/ml bovine type II collagen and 2 mg/ml Incomplete Freund’s adjuvant (IFA, Chondrex, USA). The study covered healthy control mice (n = 5), mice undergoing the disease induction protocol treated with vehicle alone that served as the positive disease control group (n = 5), and mice undergoing the disease induction protocol treated with once a week for 4 times totally of caudal-vein injection with 2 × 10 6 cells of either hUCMSCs group (CIA + hUCMSCs), ERS-hUCMSCs group (CIA + ERS-hUCMSCs) or pc-ATF4-hUCMSCs group (CIA + pc-ATF4-hUCMSCs) on day 24 after subsequent immunization with five mice in each group. MSC-based transplantation was initiated at the same time and was started following the first observable measure of disease activity as determined by disease activity score. For positive disease control group (collagen-induced arthritis, CIA), equal amount of PBS was administrated at the same time with the MSCs transplanted group through caudal vein injcetion. Mice were monitored 2–3 times each week for weight changes, paw thickness, and disease activity score at the duration of the study. Disease activity scores derived from the evaluation of clinical arthritis in all four limbs as reported by scoring system for evaluation of arthritis severity [ 17 ]. Severity score of 0–4 was determined for each limb by visual evaluation and reaching an agreement by two examiners. No evidence of erythema and swelling of the limb implied a severity score of 0, a score of 1 was assigned for erythema and mild swelling confined to the tarsals or ankle joint, a score of 2 equalled to erythema and swelling extending from the ankle to the tarsals, 3 meant erythema and extending from the ankle to metatarsal joints and a score of 4 was designated for erythema and severe swelling of the entire limb ankyloses. The disease activity score was obtained as the sum of the severity score for each limb with a peak score of 16. At experimental day 49, all the mice were euthanized using CO 2 infusion in a CO 2 line-connected box at a gas infusion rate of 1.5–3.5 L/min. Once the animals ceased respiration for 5–10 min, euthanasia was confirmed by cervical dislocation, and tissue samples were harvested for subsequent analysis. No anesthesia was used in the animal experiments of this study. The work has been reported in line with the ARRIVE guidelines 2.0. Cell transfection Small interfering RNA (siRNA) for ATF4 (GenePharma, China), OMA1 (Sevenbiotech, China) and non-targeting negative control (NC) from General Biosystems of China were transient transfected into hUCMSCs with lipofectamine 2000 reagent (Invitrogen, USA) in serum-free medium at the final concentration of 100 nM according to the manufacturer's protocol. qRT-PCR and western blot were conducted to verify the transfection effect. Cells were harvested at 24 h or 48 h following transfection for further analysis. To establish a ATF4-overexpressing hUCMSCs, a pcDNA3.1 vector containing a sequence targeting the human ATF4 gene, pcDNA3.1-ATF4 was constructed by GenePharma (China). GenePharma also provided pcDNA3.1-NC, the negative control, and lipofectamine 2000 (Invitrogen, USA) was used to transfect hUCMSCs.. After determining transfection efficiency, cells undergoing a 24 h or 48 h transfection were collected for the subsequent studies. Primer sequences are listed in table S1 . RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted from hUCMSCs utilizing TRIzol reagent (AG scientific, China) and reversed to cDNA using 5 × Master Mix II (Sevenbiotech, China) following the manufacturer's protocols. qRT-PCR was performed with the SYBR-Green Master mix (Sevenbiotech, China) in triplicate using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with the conditions of 95°C for 30 s, then 40 cycles of 95°C for 5 s and 60°C for 30 s. The RNA expression level of the target gene were normalized to the internal reference GAPDH with 2 −△△CT analysis method. The primer sequences are listed in table S2 . Immunoblotting Western blotting was performed as previously described [ 18 ]. Primary antibodies were used as following: anti-p-eIF2α (1:1000, CST, USA), anti-eIF2α (1:1000, CST, USA), anti-p-PERK (1:1000, CST, USA), anti-PERK (1:1000, CST, USA), anti-ATF4 (1:1000, CST, USA), anti-OMA1 (1:1000, CST, USA), anti-MiD49 (1:1000, Proteintech, China), anti-MiD51 (1:1000, Proteintech, China), anti-p-DRP1 (1:1000, CST, USA), anti-DRP1 (1:1000, CST, USA), anti-MFN2 (1:1000, WANLEIBIO, China), anti-OPA1 (1:1000, CST, USA), anti-Fis1 (1:1000, Affinity), anti-β-actin (1:1000, CST, USA), and anti-GAPDH (1:3000, Beyotime, China). And the fluorescent secondary antibody (1:20000, LI-COR, USA) was also employed. Odyssey CLx Infrared Scanner (Odyssey CLx, USA) was utilized to identify the results, and ImageJ software was served for calculating relative protein expression. β-actin or GAPDH served as internal references. Mitochondrial Membrane Potential Detection 3 × 10 4 cells hUCMSCs in 12-well plates were pre-incubated with or without 1µM thapsigargin (TG, Sigma, Germany) for 24 h. Mitochondrial membrane potential was assessed with the Assay Kit from AAT Bioquest (USA). Centrifuge the cells, and resuspend in the JC-10 working solution. Then the cells were incubated in a 37 ℃, 5% CO 2 incubator for 30 min. Subsequently, complete medium was added for cleaning. Finally, flow cytometry was conducted at 366 nm and 430 nm, and images were obtained through fluorescence microscope (Olympus BX53, Japan). hUCMSCs treated with FCCP (10 µM, Sigma, Germany) served as a positive control, as previously described [ 19 ]. Flow cytometry analyses for CD4 T cell subsets To detect CD4 + T cell subsets in mouse spleen and RA patient’s peripheral blood, lymphocytes were obtained from the mouse spleen after grinding and a 200-mesh filter. Peripheral blood mononuclear cells (PBMCs) from RA patients were obtained using Ficoll-Hypaque (TBD, China), and CD4 + T cells were sorted by magnetic beads (Miltenyi Biotec, Germany). Then CD4 + T cells were co-cultured with different treated hUCMSCs (20: 1) in RPMI 1640 medium (Meilunbio, China) containing anti- CD3 (2 µg/mL, eBioscience, USA) and anti-CD28 (2 µg/mL, eBioscience, USA) for 3 days. For Th1 and Th17 quantification, cells were pelleted by centrifugation and resuspended in 10% FBS RPMI-1640 medium with phorbol 12-myristate 13-acetate (200 ng/ul, Fcmacs Biotech, China) and ionomycin (1 µg/ml, Fcmacs Biotech, China) and brefeldin A (5 µg/ml, eBioscience, USA) at 37°C, 5% CO 2 for 4h. Cells were washed twice in cold PBS and stained with anti-human CD4-PE (Biolegend, USA) or anti-mouse CD4-FITC (Biolegend, USA). After permeabilization with Cytofix/Cytoperm (BD Biosciences, USA), cells were stained with anti-human IFN-γ-APC (eBioscience, USA), anti-human IL-17A-PE (eBioscience, USA) or anti-mouse IFN-γ-APC (Biolegend, USA), anti-mouse IL-17A-PE (Biolegend, USA). Tfh cells were stained with anti-human CD4-PE, anti-human PD1-PE/Cyanine7 (Biolegend, USA), anti-human CXCR5-APC (eBioscience, USA) or anti-mouse CD4-FITC, anti-mouse PD1-APC/Cyanine7 (Biolegend, USA), anti-mouse CXCR5-APC (Biolegend, USA). Histological analysis and Immunohistochemistry staining The mouse ankle joints and organs were stained as previously reported [ 20 ]. Ankles and organs were fixed, sectioned, and stained with hematoxylin-eosin (HE, Solarbio, China), Safranin O-fast green (SO/FG, Solarbio, China). Histomorphological observation was performed with a microscope (BX53, Olympus, Japan). Vimentin expression was detected through immunohistochemistry (IHC) following a series of procedures: antigen retrieval, addition of vimentin primary antibody (Proteintech, China) and IgG secondary antibody (ZSGP-BIO, China), and finally adding a detection reagent to recognize and localize the primary antibody as reported previously [ 21 ]. Statistical Analysis All statistical analyses were made by using Prism software (version 5, GraphPad). All experiments were conducted independently at least three times, and the results were expressed as mean ± standard deviation (SD). The comparison between two groups was analyzed by Student’s t-test. Statistical differences among multiple groups were analyzed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistical significance. Results ERS-hUCMSCs is more effective in alleviating joint inflammation of CIA mice than untreated hUCMSCs Since it has been verified that the immunosuppressive effect of MSCs could be enhanced by ERS-induction in vitro. Firstly, we explored the validity of ERS-MSC during RA treatment in vivo . Well-established CIA mice model was used to evaluate the effects of ERS-hUCMSCs on joint inflammation. After the onset of arthritis on day 24, the mice were injected intravenously with hUCMSCs or ERS-hUCMSCs weekly for 4 weeks (Fig. 1 A). The CIA mice gained weight more slowly than control group (Ctrl). hUCMSCs treatment (CIA + hUCMSCs) failed to result in a recovery of body weight. In contrast, ERS-hUCMSCs treatment (CIA + ERS-hUCMSCs) resulted in weight recovery to some extent (Fig. 1 B). Morphological observation of the CIA mice showed that ERS-hUCMSCs-treated mice exhibited less severe paw swelling and were ranked at lower arthritis scores (Fig. 1 C-E). Consistently, HE, SO/FG together with vimentin staining showed that ERS-hUCMSCs could effectively improve joint damage, bone erosion and synovial cell proliferation comapred to hUCMSCs-treated group (Fig. 1 F). In addition, as exhibited in Fig. 1 G-I, compared to hUCMSC-treated group, ERS-hUCMSCs exhibit stronger immunosuppressive function on major subsets of CD4 + T cells, including Th1, Th17 and Tfh derived from spleen of CIA mice. Besides, ERS-hUCMSCs decreased the frequencies of RA peripheral Th1, Th17 and Tfh subsets more obviously, compared to un-treated hUCMSCs (Fig S2 ). ATF4 is the key transcription factor regulating COX2 of ERS-hUCMSCs We have confirmed that ER-stressed MSC exhibited enhanced immunosuppressive effect through cyclooxygenase-2 (COX2) overexpression, which resulted in augmented PGE2 secretion [ 13 ]. Then we wondered the transcription factor (TF) resoposible for COX2 transcriptional regulation in ERS-hUCMSCs. Firstly, we searched TFs from TRRUST database and 58 TFs related to COX2-regulation were retrieved. Among them, ATF4 attracted our attention for its linking with cellular stress in many cell lines [ 22 ], as well as one of the signaling molecules related to ERS (Fig. 2 A left). Subsequently, we also predicted the target genes regulated by ATF4 from both TRRUST database and Cistrome Data Browser database with OmicStudio tools. It screened 41 target genes regulated by ATF4 from TRRUST database and 35 target genes were obtained after removing 6 duplicate genes. 100 target genes regulated by ATF4 were found from Cistrome Data Browser database without duplicate genes. After the intersection of the two databases, the results implied that COX2 was one of the potential target genes regulated by ATF4 (Fig. 2 A right). What's more, both mRNA and protein levels of ATF4 were elevated during ERS process (Fig 2B). Knockdown of ATF4 by specific siRNA decreased the basal levels of COX2 in the ERS condition (Fig 2C, D). Additionally, ATF4 overexpression led to enhanced COX2 mRNA expression (Fig 2E, F), which suggested that ATF4 might be a prospective TF leading to elevated COX2 expression (Fig 2G). Overexpression of ATF4 increases hUCMSCs-based immunomodulatory properties on CIA mice To verify the function of ATF4 in vivo , ATF4-overexpression hUCMSCs (pc-ATF4-hUCMSCs, 2×10 6 cells/weekly and four consecutive weeks) were transplanted to CIA mice. Morphological observation of the CIA mice indicated that pc-ATF4-hUCMSCs-treated mice exhibited more stable weight and less severe paw-swelling and were also at lower arthritis scores (Fig. 3 A-D). Besides these, HE, SO/FG and vimentin staining showed that pc-ATF4-hUCMSCs could effectively improve joint damage and bone erosion relative to hUCMSCs-treated group (Fig. 3 E). In addition, compared to hUCMSCs-treated group, pc-ATF4-hUCMSCs exhibit stronger immunosuppressive function on major subsets of CD4 + T cells, including Th1, Th17 and Tfh (Fig. 3 F-H). What's more, pc-ATF4-hUCMSCs (CD4 + T + pc-ATF4) decreased the frequencies of RA peripheral Th1, Th17 and Tfh subsets more obviously, compared to both un-treated hUCMSCs (CD4 + T + hUCMSCs) and siATF4-hUCMSCs (CD4 + T + siATF4) (Fig S3 ). PERK-mediated eIF2α phosphorylation contributes to ATF4 overexpression Since ATF4 contributed mostly to COX2-transcriptional regulation, we went on to explore the upstream of ATF4 signal axis in ERS state. Protein synthesis is controlled at several levels with translation initiation as the foremost step [23]. The ESR, when activated, gathers on the inhibition of translation initiation aiming to recover cellular homeostasis. The crucial regulatory factor is eIF2α, which phosphorylation on serine 51 causes inhibition of translation initiation, which leading us to examine eIF2α phosphorylation firstly. We found that eIF2α phosphorylation was uniquely enhanced with ERS-induction (Fig. 4 A). We went on to investigate which contributes to eIF2α phosphorylation by ERS. There are 4 different upstream kinases of eIF2α, including HRI, PKR, PERK, and GCN2 [24, 25]. And it has been verified that PERK is one of the markers involved ERS state [ 26 ]. As shown in Fig. 4 A, both PERK and eIF2α were phosphorylated during TG-induction of ERS. Additionally, GSK2606414 (GSK), a potent inhibitor of PERK, downregulated ATF4 and phosphorylation levels of eIF2α during ERS induction, implying that PERK regulated ATF4 expression probably through eIF2α phosphorylation (Fig. 4 B). To verify this conclusion, a blank control group was added. And phosphorylated eIF2α was lower than TG group after GSK treatment, but funny enough, it was yet higher than ctrl group (Fig. 4 C), illustrating that eIF2α was still activated in the condition of PERK blockage under ERS state (Fig. 4 D). ERS coupling mitochondrial stress co-regulates ATF4 expression Since ER are in close contact with mitochondria through shared mitochondria associated membranes (MAM), ER stress may be intertwined with mitochondrial function. We found that overlapping with m-AAA protease (OMA1), a major mitochondrial factor for sensing and responding to cellular stress, was in a rising trend with ER stress-time increasing (Fig. 5 A). Besides this, mitochondrial dysfunctions those closely related to mitochondrial stress were also eveluated. Mitochondrial membrane potential was tested through cellular immunofluorescence and fluorescent method. After 24 h of stimulation with TG, decreased mitochondrial membrane potential (MMP), acting as the initial signal of mitochondrial stress, could be found through JC-10 dying (Fig. 5 B). Here, FCCP served as positive control during depolarization of plasma membrane potential. Subsequently, proteins involved in mitochondrial fission (Fis1, DRP1/p-DRP1, MiD49 and MiD51) and fussion (OPA1, MFN1, MFN2) were determined during ER stress induction. The results verified that phosphorylated dynamin-related protein 1 (Drp1) on S616, mitochondrial fission 1 protein (Fis1) and mitochondrial dynamics proteins of 51 kDa (MiD51) were in down-regulation state, while mitochondrial dynamics proteins of 49 kDa (MiD49) were in increased expression. Mitochondrial fussion-related protein mitofusin-2 (MFN2) was declined. Crucially, optic atrophy−1 (OPA1) directly links mitochondrial structure and function. As illustrated in Fig. 5 C, when the transmembrane potential across the inner membrane (ΔΨm) is lost, long OPA1 isoforms (L-OPA1) is cleaved into short forms, which limited fusion and could facilitate mitochondrial fission. These data illustrated alterations in mitochondrial fission and fusion could also be detected during ER induction, implying that mitochondrial stress was also induced during TG stimulation. For further validation of the role of mitochondrial stress in the upregulation of ATF4, we transfected hUCMSCs with siRNA targeting OMA1 (Fig. 6 A). As shown in Fig. 6 B, under ERS induction, simultaneous blockade of PERK and mitochondrial stress by GSK or siOMA1, respectively have advantage over blockade alone in ATF4 downexpression, which implied that ER stress and mitochondrial stress synergistically regulated ATF4 (Fig. 6 C). Discussion Our former research demonstrated that ERS hUCMSCs possessed better inhibition effect on RA Tfh cells by releasing PGE2 [ 13 ], implying immune suppression of hUCMSCs could be enhanced by ER stress-induction. The present study demonstrated, for the first time, that ATF4 played a critical role in the enhancement of MSC-based therapy for RA through both ER and mitochondria stress. We observed that both PERK-eIF2α-ATF4 signaling pathway from ER stress and OMA1-eIF2α-ATF4 signaling pathway triggered by mitochondrial stress were involved in COX2 regulation. COX2, required for for the conversion of arachidonic acid into prostaglandins, such as PGE2, is upregulated in response to ER-stress induction and elevated COX2 will result in augmented PGE2 production, which acts as an inhibitor of T cell receptor signaling and thereby limits T cell activation [ 11 ]. It was verified in reproductive research that ATF4 bound to COX2 promoter, and COX2-derived PGE2 can regulate ovulation [ 27 ]. ATF4, a cellular stress induced-transcription factor, regulates a variety of genes involved in various physiological processes and plays a critical role as a stress-induced transcription factor. It orchestrates cellular responses, particularly in the management of ERS [ 28 , 29 ]. ER is the main organelle related to protein synthesis, modification, and processing in eukaryotic cells [ 30 ]. Mounting evidence suggests that altered ER homeostasis-events lead to the accumulation of unfolded or misfolded proteins in the ER lumen and breakdown of protein-folding homeostasis, creating a condition referred to ER stress [ 31 – 33 ]. In response to ER stress, cytoprotective axes are triggered to restore protein homeostasis in the ER through unfolded protein response (UPR), which initiates pro-survival or pro-death responses and determines cell fate via the induction of PERK, ATF6 and IRE1 pathways[ 34 ]. Besides ER, mitochondria is another important organelle in cells. Mitochondria-associated endoplasmic reticulum membranes (MAMs) are dynamic coupling structures between mitochondria and ER, which combine the two critical organelle functions. So mitochondria and ER could regulate each other via MAMs [ 35 ]. Our result manifested that ER stress could induce mitochondrial stress and eIF2α phosphorylation is the core event of the endoplasmic reticulum-mitochondria coupling stress. eIF2α phosphorylation induces the transcription factor ATF4. Moreover, mitochondrial stress is relayed to ATF4 through OMA1. Herein, we further validated that during ER-stress triggering, the induction of COX2 was in an ATF4-dependent manner and elevated COX2 is associated with cellular stress response governed by the PERK-eIF2α-ATF4 axis of the UPR pathway and OMA1-eIF2α-ATF4 of the mitochondrial stress response. Our research confirmed that the ATF4 pathway activated by both ER-stress and mitochondrial stress stimulates COX2 transcription to enhance MSC-based therapy. Conclusions Taken together, our data manifested that compared with unpre-treated hUCMSCs, ER-stressed hUCMSCs exhibited better immunosuppresive ability mainly through COX2 overexpression, which was regulated by ATF4. PERK-eIF2α activated by ERS and OMA1-eIF2α from mitochondrial stress co-regulate ATF4 expression. Abbreviations MSCs Mesenchymal stem/stromal cells ERS Endoplasmic reticulum stress RA Rheumatoid arthritis Tfh T follicular helper cells COX2 Cyclooxygenase-2 PGE2 Prostaglandin E2 TG Thapsigargin CIA Collagen-induced arthritis Th1 T helper 1 Th17 T helper 17 ATF4 Activating transcription factor 4 PERK Protein kinase RNA like endoplasmic reticulum kinase p-PERK Phosphorylated-PERK HRI Heme-Regulated Inhibitor PKR Protein Kinase R GCN2 General Control Nonderepressible 2 eIF2α Eukaryotic initiation factor 2α p-eIF2α Phosphorylated-eIF2α ER Endoplasmic reticulum UPR Unfolded protein response IRE1α Inositol requiring enzyme 1 ATF6 Activating transcription factor 6 PGE2 Prostaglandin E2 hUCMSCs Human Umbilical Cord Mesenchymal Stem Cells TF Transcription factor MAM Mitochondria associated membranes OMA1 Overlapping with m-AAA protease MMP Mitochondrial membrane potential MiD51 Mitochondrial dynamics proteins of 51 kDa MiD49 Mitochondrial dynamics proteins of 49 kDa MFN2 Mitochondrial fusion-related protein mitofusin-2 OPA1 Optic atrophy-1 L-OPA1 Long OPA1 isoforms Declarations Ethics approval and consent to participate All animal procedures were in accordance with the guidelines of the Experimental Animals Management Committee (Liaoning Province, China), and were approved by the Experimental Animals Welfare & Ethical Committee (Date:16. 05. 2024, No. AEE24013), Dalian Medical University. Project name: Research on Pathogenesis and Treatment of Autoimmune Diseases. Human MSCs and RA peripheral blood samples were obtained from consenting volunteers enrolled in this study at the Second Hospital of Dalian Medical University. This study was approved by the Ethics Committee of the Second Hospital of Dalian Medical University (Date: 08. 10. 2023, ethical approval number: 2023-253). Project name: Pathogenesis Research and Immunotherapy in Patients with Autoimmune Diseases. Consent for publication Not Applicable. Availability of data and materials All additional files are included in the manuscript. Using the OmicStudio tools, it is possible to predict the target genes regulated by ATF4 in both the TRRUST database and the Cistrome Data Browser database. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Jiaqing Liu: Formal analysis, Investigation, Writing – review & editing. Xing Zhang: Formal analysis, Investigation, Methodology, Writing – original draft. Xiangge Zhao: Methodology, Writing – review & editing. Jinyi Ren: Formal analysis, Writing – review & editing. Huina Huang and Cheng Zhang: Formal analysis, Investigation, Writing – original draft. Xianmei Chen: Writing – review & editing. Weiping Li: Human tissue providing. Jing Wei: Article designing, Writing – drafting & revising. Xia Li: Project administration, Funding acquisition, Validation. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (82071834, 82271839), Liaoning Undergraduate Program for Innovation and Entrepreneurship (S202310161043, S202310161019), Dalian key laboratory of human microorganism homeostasis and immunological mechanism research of diseases, Liaoning Provincial Education Department Basic Research Project (LJ212410161034, LJ212410161038) and Dalian Medical University Interdisciplinary Research Cooperation Project Team Funding (JCHZ2023010). The authors declare that they have not used artificial intelligence (AI)-generated work in this manuscript. The authors would like to acknowledge all lab members for insightful discussions. References Wang Y, Fang J, Liu B, Shao C, Shi Y. Reciprocal regulation of mesenchymal stem cells and immune responses. Cell Stem Cell. 2022; 29: 1515-30. Jiang B, Yao G, Tang X, Yang X, Feng X. 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Enhanced therapeutic effects of apoptotic cell-conditioned mesenchymal stem cells in lupus-prone MRL/lpr mice. Rheumatol & Autoimmun. 2024; 4: 90-8. Chen X, Cubillos-Ruiz JR.Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat Rev Cancer. 2021; 21: 71-88. Marciniak SJ, Chambers JE, Ron D. Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov. 2022; 21: 115-40. Chen Z, Tian R, She Z, Cai J, Li H. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. Free Radic Biol Med. 2020; 152: 116-41. Pandey VK, Mathur A, Khan MF, Kakkar P. Activation of PERK-eIF2α-ATF4 pathway contributes to diabetic hepatotoxicity: Attenuation of ER stress by Morin. Cell Signal. 2019; 59: 41-52. Wang M, Xue Y, Du F, Ma L, Lu LJ, Jiang L, et al.Continuation, reduction, or withdrawal of tofacitinib in patients with rheumatoid arthritis achieving sustained disease control: a multicenter, open-label, randomized controlled trial. Chin Med J (Engl). 2023; 136: 331-40. Wei J, Ouyang X, Tang Y, Li H, Wang B, Ye Y, et al. ER-stressed MSC displayed more effective immunomodulation in RA CD4(+)CXCR5(+)ICOS(+) follicular helper-like T cells through higher PGE2 binding with EP2/EP4. Mod Rheumatol 2020; 30: 509-16. de Seabra Rodrigues Dias IR, Lo HH, Zhang K, Law BYK, Nasim AA, Chung SK, et al. Potential therapeutic compounds from traditional Chinese medicine targeting endoplasmic reticulum stress to alleviate rheumatoid arthritis. Pharmacol Res. 2021; 170: 105696. Zhao C, Zhang L, Kong W, Liang J, Xu X, Wu H, et al. Umbilical Cord-Derived Mesenchymal Stem Cells Inhibit Cadherin-11 Expression by Fibroblast-Like Synoviocytes in Rheumatoid Arthritis. J Immunol Res. 2015; 2015: 137695. Xiang E, Han B, Zhang Q, Rao W, Wang Z, Chang C, et al. Human umbilical cord-derived mesenchymal stem cells prevent the progression of early diabetic nephropathy through inhibiting inflammation and fibrosis. Stem Cell Res Ther. 2020; 11: 336. Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc, 2007; 2: 1269-75 Wei J, Huang X, Zhang X, Chen G, Zhang C, Zhou X, et al. Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway. Cell Death Dis. 2023;14:97. Klier PEZ, Martin JG, Miller EW. Imaging reversible mitochondrial membrane potential dynamics with a masked rhodamine voltage reporter. J Am Chem Soc. 2021; 143: 4095-9. Lin WW, Ho KW, Su HH, Fang TF, Tzou SC, Chen IJ, et al. Fibrinogen-Like protein 1 serves as an anti-inflammatory agent for collagen-induced arthritis therapy in mice. Front Immunol. 2021;12: 767868. Magaki S, Hojat SA, Wei B, So A, Yong WH. An Introduction to the Performance of Immunohistochemistry.Methods Mol Biol. 2019;1897: 289-98. Wortel IMN, van der Meer LT, Kilberg MS, van Leeuwen FN. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells. Trends Endocrinol Metab. 2017; 28: 794-806. Lomakin IB, Steitz TA. The initiation of mammalian protein synthesis and mRNA scanning mechanism. Nature. 2013; 500: 307-11. Wen W, Zhao Q, Yin M, Qin L, Hu J, Chen H, et al. Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction. Front Immunol. 2020; 11: 577838. Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT, et al. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature. 2020; 579: 427-32. Zeng T, Zhou Y, Yu Y, Wang JW, Wu Y, Wang X, et al. rmMANF prevents sepsis-associated lung injury via inhibiting endoplasmic reticulum stress-induced ferroptosis in mice. Int Immunopharmacol. 2023;114:109608. Di F, Liu J, Li S, Yao G, Hong Y, Chen ZJ, et al. ATF4 Contributes to Ovulation via Regulating COX2/PGE2 Expression: A Potential Role of ATF4 in PCOS. Front Endocrinol (Lausanne). 2018; 9: 669 Tang H, Kang R, Liu J, Tang D. ATF4 in cellular stress, ferroptosis, and cancer. Arch Toxicol. 2024; 98: 1025-41. Neill G, Masson GR. A stay of execution: ATF4 regulation and potential outcomes for the integrated stress response. Front Mol Neurosci. 2023; 16: 1112253. Hughes A, Oxford AE, Tawara K, Jorcyk CL, Oxford JT. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis. Int J Mol Sci. 2017; 18: 665. Hetz C, Papa FR. The Unfolded Protein Response and Cell Fate Control. Mol Cell. 2018; 69: 169-81. Hetz C, Chevet E, Oakes SA. Proteostasis control by the unfolded protein response. Nat Cell Biol. 2015; 17: 829-38. Karagoz GE, Acosta-Alvear D, Walter P. The Unfolded Protein Response: Detecting and Responding to Fluctuations in the Protein-Folding Capacity of the Endoplasmic Reticulum. Cold Spring Harb Perspect Biol. 2019; 11: a033886. Kasahara A, Scorrano L. Mitochondria: from cell death executioners to regulators of cell differentiation. Trends Cell Biol. 2014; 24: 761-70. He Q, Qu M, Shen T, Su J, Xu Y, Xu C, et al. Control of mitochondria-associated endoplasmic reticulum membranes by protein S-palmitoylation: Novel therapeutic targets for neurodegenerative diseases. Ageing Res Rev. 2023; 87: 101920. Supplementary Files AuthorChecklistFull.pdf SupplementaryFigures.pdf Cite Share Download PDF Status: Published Journal Publication published 28 May, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 21 Jan, 2025 Reviewers invited by journal 21 Jan, 2025 Editor assigned by journal 15 Jan, 2025 First submitted to journal 08 Jan, 2025 Editorial decision: Major Revision 17 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5620379","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":405261820,"identity":"7a1d866c-8e12-452a-abc9-ed3aef9b460a","order_by":0,"name":"Jiaqing Liu","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqing","middleName":"","lastName":"Liu","suffix":""},{"id":405261821,"identity":"bf54ddee-e2eb-455a-b0a0-c872201e22a6","order_by":1,"name":"Xing Zhang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Zhang","suffix":""},{"id":405261822,"identity":"0fd06e2c-8ced-4b47-8c02-fc70a6db3767","order_by":2,"name":"Xiangge Zhao","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiangge","middleName":"","lastName":"Zhao","suffix":""},{"id":405261823,"identity":"87304339-5d44-499c-9f6e-504a3c3e0019","order_by":3,"name":"Jinyi Ren","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinyi","middleName":"","lastName":"Ren","suffix":""},{"id":405261824,"identity":"01e930b4-7bf2-42aa-9702-087c7dc58ec7","order_by":4,"name":"Huina Huang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huina","middleName":"","lastName":"Huang","suffix":""},{"id":405261825,"identity":"3980c163-d787-4d17-8658-54afeae18b40","order_by":5,"name":"Cheng Zhang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhang","suffix":""},{"id":405261826,"identity":"4083452b-64b6-40e8-889a-07998b058bde","order_by":6,"name":"Xianmei Chen","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianmei","middleName":"","lastName":"Chen","suffix":""},{"id":405261827,"identity":"d3c45494-6a94-4d0a-8c96-1b7f8e42f979","order_by":7,"name":"Weiping Li","email":"","orcid":"","institution":"The Second Hospital of Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiping","middleName":"","lastName":"Li","suffix":""},{"id":405261828,"identity":"953d1423-8a1f-4298-a8c9-8e21d0821400","order_by":8,"name":"Jing Wei","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wei","suffix":""},{"id":405261829,"identity":"2cfec0b2-333d-48b6-8d69-3e869492ee35","order_by":9,"name":"Xia li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACPmYGhgMMPBJy/MzMhx8QpYUNrEXGwliynS3NgDgtYNKmItHgPI+CBHFa2HkMD/zIkUgwPszDYMBQYxNNhMPYEg72nJHIMzvMe+ABw7G03AbCWpgPHODtkSg2O8yXYMDYcJgYLYwNB//+k0jc3MxjIEGkFuYDh3l4JBI3MBOvhS3hsAyPhLHEYWAgJxDjF37+M8Yf3/DUyfH3Hz784EONDWEtqCCBNOWjYBSMglEwCnABAFFRNyipJg+UAAAAAElFTkSuQmCC","orcid":"","institution":"Dalian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xia","middleName":"","lastName":"li","suffix":""}],"badges":[],"createdAt":"2024-12-11 02:54:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5620379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5620379/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04362-x","type":"published","date":"2025-05-28T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74587205,"identity":"123c2499-92da-4203-bc0b-9e4553c53964","added_by":"auto","created_at":"2025-01-23 16:55:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransplantation of ERS-hUCMSCs improved therapeutic efficacy compared to hUCMSCs in a mouse model of CIA. \u003c/strong\u003eFor ERS induction hUCMSCs were treated with TG (1 μM, 6 h). \u003cstrong\u003eA. \u003c/strong\u003eThe flowchart of CIA mice establishment and treatment from day 0 to day 49 (n=5). \u003cstrong\u003eB. \u003c/strong\u003eThe growth curve of body weight (n=5). \u003cstrong\u003eC. \u003c/strong\u003eChange in paw thickness curves (n=5). \u003cstrong\u003eD. \u003c/strong\u003eClinical scoring of arthritis symptoms in CIA (n=5). \u003cstrong\u003eE. \u003c/strong\u003ePictures of representative paws taken at the end of the experiment (n=5). \u003cstrong\u003eF.\u003c/strong\u003e Representative picutres of ankle joint histopathological changes shown by HE staining, SO/FG staining and immunohistochemistry (IHC) staining of vimentin (n=5). \u003cstrong\u003eG-I.\u003c/strong\u003e Flow cytometry analysis of activated signature markers of Th1, Th17, and Tfh (IFN-γ, IL-17A, PD-1 and CXCR5) on the range of CD4⁺ T cells isolated from the spleens of indicated group (n=5), detected on the terminal day after the induction of CIA. The mean values are expressed as ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/5844e450e391d38e63b284c1.png"},{"id":74587573,"identity":"eb7a9b09-6752-4c6a-8daa-e12b0c020e9c","added_by":"auto","created_at":"2025-01-23 17:03:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF4 positively regulates COX2 in ERS-hUCMSCs. A. \u003c/strong\u003eTRRUST and Cistrome Data Browser databases were used to analyze transcription factors involved in regulation of COX2 and target genes involved in ATF4-regulation. \u003cstrong\u003eB. \u003c/strong\u003emRNA and protein levels of ATF4 in TG treated or un-treated groups (n=3). Full-length blots are presented in Supplementary Figure 4A. \u003cstrong\u003eC-F.\u003c/strong\u003emRNA levels of COX2 in hUCMSCs transfected with either ATF4 knockdown or ATF4 overexpression. \u003cstrong\u003eC.\u003c/strong\u003e mRNA and protein levels of ATF4 following transfection with different siRNAs by qRT-PCR and westernblot for 24 h and 48 h, respectively. siRNA, small interfering RNA; NC, negative control (n=3).\u003cstrong\u003e \u003c/strong\u003eFull-length blots are presented in Supplementary Figure 4B.\u003cstrong\u003e \u0026nbsp;D. \u003c/strong\u003emRNA levels of COX2 under TG-stimulation in ATF4 knockdown (n=3). \u003cstrong\u003eE.\u003c/strong\u003e mRNA and protein levels of ATF4 following transfection with overexpressed plasmid by qRT-PCR and westernblot for 24 h and 48 h, respectively. pc-ATF4, plasmid construct-ATF4; NC, negative control (n=3).\u003cstrong\u003e \u003c/strong\u003eFull-length blots are presented in Supplementary Figure 4C. \u003cstrong\u003eF. \u003c/strong\u003emRNA levels of COX2 under TG-stimulation or ATF4 overexpression (n=3). \u003cstrong\u003eG. \u003c/strong\u003eERS-hUCMSCs up-regulated COX2 expression through ATF4. Data are means ± SD of three independent experiments, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/bb9391f8cb03b99ba35fea6a.png"},{"id":74587215,"identity":"0c94c70d-f324-403e-9a81-a927dabac2a0","added_by":"auto","created_at":"2025-01-23 16:55:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":769328,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effects of pc-ATF4-hUCMSCs transplantation in CIA mice.\u003c/p\u003e\n\u003cp\u003eA decrease in severity of CIA following pc-ATF4-hUCMSCs transplantation is shown by body weight (\u003cstrong\u003eA\u003c/strong\u003e, n=5) paw thickness (\u003cstrong\u003eB\u003c/strong\u003e, n=5) and arthritis index scores (\u003cstrong\u003eC\u003c/strong\u003e, n=5). Pictures of representative paws swelling (\u003cstrong\u003eD\u003c/strong\u003e), HE and SO/FG stainings and subsequent histological examination of vimentin (\u003cstrong\u003eE\u003c/strong\u003e) were performed at the end of the experiment (n=5). \u003cstrong\u003eF-H. \u003c/strong\u003eDetections of CD4\u003csup\u003e+\u003c/sup\u003e T subsets , including Th1 (F), Th17 (G), and Tfh (H) subpopulations in the spleens of mice from indicated group (n=5). The mean values are expressed as ± SD, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/a0c516171ad01d123f5a3eac.png"},{"id":74587204,"identity":"178df64d-a86d-4124-a904-665c5c7bf060","added_by":"auto","created_at":"2025-01-23 16:55:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":272839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePERK mediated elf2α phosphorylation in hUCMSCs induced by ERS is necessary for ATF4 Up-regulation. A.\u003c/strong\u003e Representative western blots using antibodies targeting p-elf2α, total elf2α, p-PERK, total PERK, and β-actin (n=3). Full-length blots are presented in Supplementary Figure 5A. \u003cstrong\u003eB. \u003c/strong\u003eATF4, p-elf2α, total elf2α, and β-actin protein levels of ERS-hUCMSCs in the presence or absence (control medium) of the PERK inhibitor, GSK2606414 (GSK, 1 µM, n=3). Full-length blots are presented in Supplementary Figure 5B. \u003cstrong\u003eC. \u003c/strong\u003eProtein levels of p-PERK, total PERK, p-elf2α, total elf2α and β-actin in ERS-hUCMSCs with or without GSK (n=3). Full-length blots are presented in Supplementary Figure 5C. Protein bands intensity was quantified by ImageJ. \u003cstrong\u003eD. \u003c/strong\u003eSchematic diagram of the specific mechanism of ATF4 regulated by ERS. Data are means ± SD of three independent experiments, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/3449d85a76ed5b707fa5760b.png"},{"id":74587209,"identity":"c9d1b3a5-4b28-404d-9f76-69c7ab62b18a","added_by":"auto","created_at":"2025-01-23 16:55:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial stress was evoked in ERS-hUCMSCs. A.\u003c/strong\u003e Protein levels of OMA1 from control and TG-treated hUCMSCs in a time-dependent manner (n=3). Full-length blots are presented in Supplementary Figure 6A. \u003cstrong\u003eB.\u003c/strong\u003e Observation of cell mitochondrial membrane potential by fluorescence and flow cytometry (n=4). \u003cstrong\u003eC.\u003c/strong\u003e Protein levels of mitochondrial dynamics related proteins. The levels of the mitochondrial fission proteins, Fis1, MiD49, MiD51, p-Drp1and Drp1 (left), and the mitochondrial fusion proteins, Opa1 and Mfn2 (right), were identified via western blot analysis (n=3). Protein levels in each group are relative to the control group. Full-length blots are presented in Supplementary Figure 6B. All values are shown as the means ± SD from three independent studies. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/a8466dede30762a687c1ae82.png"},{"id":74588389,"identity":"3ac4001f-5c01-46bc-90aa-96fdbbc59140","added_by":"auto","created_at":"2025-01-23 17:11:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":184886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERS-integrated mitochondrial stress synergistically regulated ATF4. A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInterference effect of OMA1 from both mRNA (left) and protein (right) levels (n=3). Full-length blots are presented in Supplementary Figure 7A. \u003cstrong\u003eB.\u003c/strong\u003e Western blot analysis of p-elf2α, elf2α and ATF4 following hUCMSCs were transfected with siRNA targeting OMA1 and treated with GSK or TG (n=3). Full-length blots are presented in Supplementary Figure 7B. Data are means ± SD of three independent experiments, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/3d585a6b9d8e31faf96703c0.png"},{"id":74587231,"identity":"29c1d44f-1734-4bfd-8902-c0d15b27e4a7","added_by":"auto","created_at":"2025-01-23 16:55:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":122454,"visible":true,"origin":"","legend":"\u003cp\u003eA proposed model of how ERS enhances the therapeutic effect of MSCs during RA treatment. ERS and mitochondrial stress co-activates ATF4 in hUCMSCs through eIF2α phosphorylation.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/d3420db51eff11c4a4d0413f.png"},{"id":83783437,"identity":"6da06bc6-5311-44df-b422-898efe657155","added_by":"auto","created_at":"2025-06-02 16:11:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3822909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/6cebae5c-6529-406b-9714-23bec5a8248d.pdf"},{"id":74587202,"identity":"9cd19743-d594-46be-b2b1-05808dd77982","added_by":"auto","created_at":"2025-01-23 16:55:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":290349,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/efaf4d1f4036ce07e006d41a.pdf"},{"id":74587206,"identity":"3b8abc42-2917-405f-9088-827f1fbd825c","added_by":"auto","created_at":"2025-01-23 16:55:04","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":454993,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5620379/v1/dbc6811409ce7f1a400f20da.pdf"}],"financialInterests":"","formattedTitle":"Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapy for rheumatoid arthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMesenchymal stromal/stem cells (MSCs) have been serving as a valuable source for treatment of immune-mediated disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MSC-based therapies have encountered translational hurdles for the decreased immunosuppressive capacities post delivery [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Researchers are interested in developing new strategies to increase the immunosuppressive potential of MSCs in clinical applications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Nowadays, many approaches have been proposed to enhance the therapeutic effects of MSCs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among them, pre-treated MSCs are easier to manipulate and have been studied in a variety of disease models [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded or misfolded proteins in ER can lead to ER stress. ER stress functions as a double-edged sword, leading to apoptosis or resulting in cellular function changes, which determine cell function, fate and survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Disturbed ER homeostasis activates unfolded protein response (UPR) sensor proteins, including protein kinase RNA like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1 (IRE1α) and activating transcription factor 6 (ATF6) to restore the equilibrium. Activated PERK phosphorylates eukaryotic initiation factor 2α (eIF2α) which prevents translation of most mRNAs by inhibiting the initiation complex [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Concurrently, phosphorylation of eIF2α favors increased translation of selective mRNAs, and one of the increased protein-translations is activating transcription factor 4 (ATF4), a principal regulator which plays a crucial role in the adaptation to stresses through regulating the transcription of many genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRheumatoid arthritis (RA) is a progressive and chronic autoimmune disease. Although the exact causes are unknown, the differentiation of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes into pathogenic CD4\u003csup\u003e+\u003c/sup\u003e T subsets occupies a remarkable position in RA pathogenesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Some RA therapeutic medications have displayed ER stress-modulating properties. It has been verified that ER stress-modulating traditional Chinese medicines as potential new pharmaceutical drugs for RA clinical therapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our previous results have shown that ER-stressed (ERS) MSCs displayed better immunomodulation effects in Tfh subsets from RA patients through elevated prostaglandin E2 (PGE2) secretion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This study aimed at exploring the therapeutic effect of ERS-MSC on ameliorating the severity of arthritis in a mouse model of RA, together with the molecular mechanism of PGE2 higher expression, which could provide preclinical experimental data for the development of new stem cell drugs for the treatment of RA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation, culture, and identification of hUCMSCs\u003c/h2\u003e \u003cp\u003eHuman Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) were isolated from healthy donors who gave birth and signed informed consents according to protocols reported previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The umbilical cord tissues were cut into small segments of about 1.0 mm3, then washed with PBS and centrifuged at 1900 r/min for 6 min. And cultured in an incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e concentration with DMEM/F12 (Meilunbio, China) consisting of 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA), 100 U/mL penicillin (Solarbio, China), and 100 mg/mL streptomycin (Solarbio, China). The primary cells were harvested when the confluence reached 80% and further characterized by flow cytometry (Agilent, USA) with following fluorescein-labeled antibodies: CD90-Perep, CD105-PE, CD45-FITC, CD34-APC, CD14-FITC, CD11b-APC, and HLA-DR-PE (eBioscience, USA). Isotype-matched control antibodies were used as methodology controls. After identification (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), hUCMSCs were treated with TG for ERS induction and followed with verificaion of an ERS induction (data not shown), as mentioned previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cells reaching the fifth passage were employed in the following experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals, disease induction and treatment\u003c/h3\u003e\n\u003cp\u003eMale adult DBA/1 mice at 6\u0026ndash;8 weeks were purchased from Shanghai SLAC Laboratory Animals Company and housed under pathogen-free conditions at the Laboratory Animal Center of Dalian Medical University. Following a one-week acclimation period, mice undergoing the disease induction protocol were administered intradermal tail injections of a prepared collagen/Complete Freund adjuvant (CFA, Chondrex, USA) emulsion containing 1 mg/ml bovine type II collagen (Chondrex, USA) and 2 mg/ml at experimental day 0. Mice received a subsequent intradermal tail booster injection on day 21 with emulsion containing 1 mg/ml bovine type II collagen and 2 mg/ml Incomplete Freund\u0026rsquo;s adjuvant (IFA, Chondrex, USA).\u003c/p\u003e \u003cp\u003eThe study covered healthy control mice (n\u0026thinsp;=\u0026thinsp;5), mice undergoing the disease induction protocol treated with vehicle alone that served as the positive disease control group (n\u0026thinsp;=\u0026thinsp;5), and mice undergoing the disease induction protocol treated with once a week for 4 times totally of caudal-vein injection with 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells of either hUCMSCs group (CIA\u0026thinsp;+\u0026thinsp;hUCMSCs), ERS-hUCMSCs group (CIA\u0026thinsp;+\u0026thinsp;ERS-hUCMSCs) or pc-ATF4-hUCMSCs group (CIA\u0026thinsp;+\u0026thinsp;pc-ATF4-hUCMSCs) on day 24 after subsequent immunization with five mice in each group. MSC-based transplantation was initiated at the same time and was started following the first observable measure of disease activity as determined by disease activity score. For positive disease control group (collagen-induced arthritis, CIA), equal amount of PBS was administrated at the same time with the MSCs transplanted group through caudal vein injcetion. Mice were monitored 2\u0026ndash;3 times each week for weight changes, paw thickness, and disease activity score at the duration of the study.\u003c/p\u003e \u003cp\u003eDisease activity scores derived from the evaluation of clinical arthritis in all four limbs as reported by scoring system for evaluation of arthritis severity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Severity score of 0\u0026ndash;4 was determined for each limb by visual evaluation and reaching an agreement by two examiners. No evidence of erythema and swelling of the limb implied a severity score of 0, a score of 1 was assigned for erythema and mild swelling confined to the tarsals or ankle joint, a score of 2 equalled to erythema and swelling extending from the ankle to the tarsals, 3 meant erythema and extending from the ankle to metatarsal joints and a score of 4 was designated for erythema and severe swelling of the entire limb ankyloses. The disease activity score was obtained as the sum of the severity score for each limb with a peak score of 16. At experimental day 49, all the mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e infusion in a CO\u003csub\u003e2\u003c/sub\u003e line-connected box at a gas infusion rate of 1.5\u0026ndash;3.5 L/min. Once the animals ceased respiration for 5\u0026ndash;10 min, euthanasia was confirmed by cervical dislocation, and tissue samples were harvested for subsequent analysis. No anesthesia was used in the animal experiments of this study. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003ch3\u003eCell transfection\u003c/h3\u003e\n\u003cp\u003eSmall interfering RNA (siRNA) for ATF4 (GenePharma, China), OMA1 (Sevenbiotech, China) and non-targeting negative control (NC) from General Biosystems of China were transient transfected into hUCMSCs with lipofectamine 2000 reagent (Invitrogen, USA) in serum-free medium at the final concentration of 100 nM according to the manufacturer's protocol. qRT-PCR and western blot were conducted to verify the transfection effect. Cells were harvested at 24 h or 48 h following transfection for further analysis.\u003c/p\u003e \u003cp\u003eTo establish a ATF4-overexpressing hUCMSCs, a pcDNA3.1 vector containing a sequence targeting the human ATF4 gene, pcDNA3.1-ATF4 was constructed by GenePharma (China). GenePharma also provided pcDNA3.1-NC, the negative control, and lipofectamine 2000 (Invitrogen, USA) was used to transfect hUCMSCs.. After determining transfection efficiency, cells undergoing a 24 h or 48 h transfection were collected for the subsequent studies. Primer sequences are listed in table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA Isolation and Quantitative Real-Time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from hUCMSCs utilizing TRIzol reagent (AG scientific, China) and reversed to cDNA using 5 \u0026times; Master Mix II (Sevenbiotech, China) following the manufacturer's protocols. qRT-PCR was performed with the SYBR-Green Master mix (Sevenbiotech, China) in triplicate using the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with the conditions of 95\u0026deg;C for 30 s, then 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. The RNA expression level of the target gene were normalized to the internal reference GAPDH with 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e analysis method. The primer sequences are listed in table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eWestern blotting was performed as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Primary antibodies were used as following: anti-p-eIF2α (1:1000, CST, USA), anti-eIF2α (1:1000, CST, USA), anti-p-PERK (1:1000, CST, USA), anti-PERK (1:1000, CST, USA), anti-ATF4 (1:1000, CST, USA), anti-OMA1 (1:1000, CST, USA), anti-MiD49 (1:1000, Proteintech, China), anti-MiD51 (1:1000, Proteintech, China), anti-p-DRP1 (1:1000, CST, USA), anti-DRP1 (1:1000, CST, USA), anti-MFN2 (1:1000, WANLEIBIO, China), anti-OPA1 (1:1000, CST, USA), anti-Fis1 (1:1000, Affinity), anti-β-actin (1:1000, CST, USA), and anti-GAPDH (1:3000, Beyotime, China). And the fluorescent secondary antibody (1:20000, LI-COR, USA) was also employed. Odyssey CLx Infrared Scanner (Odyssey CLx, USA) was utilized to identify the results, and ImageJ software was served for calculating relative protein expression. β-actin or GAPDH served as internal references.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential Detection\u003c/h2\u003e \u003cp\u003e3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells hUCMSCs in 12-well plates were pre-incubated with or without 1\u0026micro;M thapsigargin (TG, Sigma, Germany) for 24 h. Mitochondrial membrane potential was assessed with the Assay Kit from AAT Bioquest (USA). Centrifuge the cells, and resuspend in the JC-10 working solution. Then the cells were incubated in a 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 30 min. Subsequently, complete medium was added for cleaning. Finally, flow cytometry was conducted at 366 nm and 430 nm, and images were obtained through fluorescence microscope (Olympus BX53, Japan). hUCMSCs treated with FCCP (10 \u0026micro;M, Sigma, Germany) served as a positive control, as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow cytometry analyses for CD4 T cell subsets\u003c/h3\u003e\n\u003cp\u003eTo detect CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets in mouse spleen and RA patient\u0026rsquo;s peripheral blood, lymphocytes were obtained from the mouse spleen after grinding and a 200-mesh filter. Peripheral blood mononuclear cells (PBMCs) from RA patients were obtained using Ficoll-Hypaque (TBD, China), and CD4\u003csup\u003e+\u003c/sup\u003e T cells were sorted by magnetic beads (Miltenyi Biotec, Germany). Then CD4\u003csup\u003e+\u003c/sup\u003e T cells were co-cultured with different treated hUCMSCs (20: 1) in RPMI 1640 medium (Meilunbio, China) containing anti- CD3 (2 \u0026micro;g/mL, eBioscience, USA) and anti-CD28 (2 \u0026micro;g/mL, eBioscience, USA) for 3 days.\u003c/p\u003e \u003cp\u003eFor Th1 and Th17 quantification, cells were pelleted by centrifugation and resuspended in 10% FBS RPMI-1640 medium with phorbol 12-myristate 13-acetate (200 ng/ul, Fcmacs Biotech, China) and ionomycin (1 \u0026micro;g/ml, Fcmacs Biotech, China) and brefeldin A (5 \u0026micro;g/ml, eBioscience, USA) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 4h. Cells were washed twice in cold PBS and stained with anti-human CD4-PE (Biolegend, USA) or anti-mouse CD4-FITC (Biolegend, USA). After permeabilization with Cytofix/Cytoperm (BD Biosciences, USA), cells were stained with anti-human IFN-γ-APC (eBioscience, USA), anti-human IL-17A-PE (eBioscience, USA) or anti-mouse IFN-γ-APC (Biolegend, USA), anti-mouse IL-17A-PE (Biolegend, USA). Tfh cells were stained with anti-human CD4-PE, anti-human PD1-PE/Cyanine7 (Biolegend, USA), anti-human CXCR5-APC (eBioscience, USA) or anti-mouse CD4-FITC, anti-mouse PD1-APC/Cyanine7 (Biolegend, USA), anti-mouse CXCR5-APC (Biolegend, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological analysis and Immunohistochemistry staining\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe mouse ankle joints and organs were stained as previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Ankles and organs were fixed, sectioned, and stained with hematoxylin-eosin (HE, Solarbio, China), Safranin O-fast green (SO/FG, Solarbio, China). Histomorphological observation was performed with a microscope (BX53, Olympus, Japan). Vimentin expression was detected through immunohistochemistry (IHC) following a series of procedures: antigen retrieval, addition of vimentin primary antibody (Proteintech, China) and IgG secondary antibody (ZSGP-BIO, China), and finally adding a detection reagent to recognize and localize the primary antibody as reported previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were made by using Prism software (version 5, GraphPad). All experiments were conducted independently at least three times, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The comparison between two groups was analyzed by Student\u0026rsquo;s t-test. Statistical differences among multiple groups were analyzed by one-way analysis of variance (ANOVA). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eERS-hUCMSCs is more effective in alleviating joint inflammation of CIA mice than untreated hUCMSCs\u003c/h2\u003e\n \u003cp\u003eSince it has been verified that the immunosuppressive effect of MSCs could be enhanced by ERS-induction \u003cem\u003ein vitro.\u003c/em\u003e Firstly, we explored the validity of ERS-MSC during RA treatment \u003cem\u003ein vivo\u003c/em\u003e. Well-established CIA mice model was used to evaluate the effects of ERS-hUCMSCs on joint inflammation. After the onset of arthritis on day 24, the mice were injected intravenously with hUCMSCs or ERS-hUCMSCs weekly for 4 weeks (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The CIA mice gained weight more slowly than control group (Ctrl). hUCMSCs treatment (CIA\u0026thinsp;+\u0026thinsp;hUCMSCs) failed to result in a recovery of body weight. In contrast, ERS-hUCMSCs treatment (CIA\u0026thinsp;+\u0026thinsp;ERS-hUCMSCs) resulted in weight recovery to some extent (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Morphological observation of the CIA mice showed that ERS-hUCMSCs-treated mice exhibited less severe paw swelling and were ranked at lower arthritis scores (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). Consistently, HE, SO/FG together with vimentin staining showed that ERS-hUCMSCs could effectively improve joint damage, bone erosion and synovial cell proliferation comapred to hUCMSCs-treated group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, as exhibited in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG-I, compared to hUCMSC-treated group, ERS-hUCMSCs exhibit stronger immunosuppressive function on major subsets of CD4\u003csup\u003e+\u003c/sup\u003e T cells, including Th1, Th17 and Tfh derived from spleen of CIA mice. Besides, ERS-hUCMSCs decreased the frequencies of RA peripheral Th1, Th17 and Tfh subsets more obviously, compared to un-treated hUCMSCs (Fig \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eATF4 is the key transcription factor regulating COX2 of ERS-hUCMSCs\u003c/h2\u003e\n \u003cp\u003eWe have confirmed that ER-stressed MSC exhibited enhanced immunosuppressive effect through cyclooxygenase-2 (COX2) overexpression, which resulted in augmented PGE2 secretion [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Then we wondered the transcription factor (TF) resoposible for COX2 transcriptional regulation in ERS-hUCMSCs. Firstly, we searched TFs from TRRUST database and 58 TFs related to COX2-regulation were retrieved. Among them, ATF4 attracted our attention for its linking with cellular stress in many cell lines [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], as well as one of the signaling molecules related to ERS (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA left).\u003c/p\u003e\n \u003cp\u003eSubsequently, we also predicted the target genes regulated by ATF4 from both TRRUST database and Cistrome Data Browser database with OmicStudio tools. It screened 41 target genes regulated by ATF4 from TRRUST database and 35 target genes were obtained after removing 6 duplicate genes. 100 target genes regulated by ATF4 were found from Cistrome Data Browser database without duplicate genes. After the intersection of the two databases, the results implied that COX2 was one of the potential target genes regulated by ATF4 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA right).\u003c/p\u003e\n \u003cp\u003eWhat\u0026apos;s more, both mRNA and protein levels of ATF4 were elevated during ERS \u0026nbsp;process (Fig 2B). Knockdown of ATF4 by specific siRNA decreased the basal levels of COX2 in the ERS condition (Fig 2C, D). Additionally, ATF4 overexpression led to enhanced COX2 mRNA expression (Fig 2E, F), which suggested that ATF4 might be a prospective TF leading to elevated COX2 expression (Fig 2G).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eOverexpression of ATF4 increases hUCMSCs-based immunomodulatory properties on CIA mice\u003c/h2\u003e\n \u003cp\u003eTo verify the function of ATF4 \u003cem\u003ein vivo\u003c/em\u003e, ATF4-overexpression hUCMSCs (pc-ATF4-hUCMSCs, 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/weekly and four consecutive weeks) were transplanted to CIA mice. Morphological observation of the CIA mice indicated that pc-ATF4-hUCMSCs-treated mice exhibited more stable weight and less severe paw-swelling and were also at lower arthritis scores (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Besides these, HE, SO/FG and vimentin staining showed that pc-ATF4-hUCMSCs could effectively improve joint damage and bone erosion relative to hUCMSCs-treated group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, compared to hUCMSCs-treated group, pc-ATF4-hUCMSCs exhibit stronger immunosuppressive function on major subsets of CD4\u003csup\u003e+\u003c/sup\u003e T cells, including Th1, Th17 and Tfh (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). What\u0026apos;s more, pc-ATF4-hUCMSCs (CD4\u003csup\u003e+\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;pc-ATF4) decreased the frequencies of RA peripheral Th1, Th17 and Tfh subsets more obviously, compared to both un-treated hUCMSCs (CD4\u003csup\u003e+\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;hUCMSCs) and siATF4-hUCMSCs (CD4\u003csup\u003e+\u003c/sup\u003e T\u0026thinsp;+\u0026thinsp;siATF4) (Fig \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003ePERK-mediated eIF2\u0026alpha; phosphorylation contributes to ATF4 overexpression\u003c/h2\u003e\n \u003cp\u003eSince ATF4 contributed mostly to COX2-transcriptional regulation, we went on to explore the upstream of ATF4 signal axis in ERS state. Protein synthesis is controlled at several levels with translation initiation as the foremost step [23]. The ESR, when activated, gathers on the inhibition of translation initiation aiming to recover cellular homeostasis. The crucial regulatory factor is eIF2\u0026alpha;, which phosphorylation on serine 51 causes inhibition of translation initiation, which leading us to examine eIF2\u0026alpha; phosphorylation firstly. We found that eIF2\u0026alpha; phosphorylation was uniquely enhanced with ERS-induction (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eWe went on to investigate which contributes to eIF2\u0026alpha; phosphorylation by ERS. There are 4 different upstream kinases of eIF2\u0026alpha;, including HRI, PKR, PERK, and GCN2 [24, 25]. And it has been verified that PERK is one of the markers involved ERS state [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, both PERK and eIF2\u0026alpha; were phosphorylated during TG-induction of ERS. Additionally, GSK2606414 (GSK), a potent inhibitor of PERK, downregulated ATF4 and phosphorylation levels of eIF2\u0026alpha; during ERS induction, implying that PERK regulated ATF4 expression probably through eIF2\u0026alpha; phosphorylation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). To verify this conclusion, a blank control group was added. And phosphorylated eIF2\u0026alpha; was lower than TG group after GSK treatment, but funny enough, it was yet higher than ctrl group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), illustrating that eIF2\u0026alpha; was still activated in the condition of PERK blockage under ERS state (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eERS coupling mitochondrial stress co-regulates ATF4 expression\u003c/h2\u003e\n \u003cp\u003eSince ER are in close contact with mitochondria through shared mitochondria associated membranes (MAM), ER stress may be intertwined with mitochondrial function. We found that overlapping with m-AAA protease (OMA1), a major mitochondrial factor for sensing and responding to cellular stress, was in a rising trend with ER stress-time increasing (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Besides this, mitochondrial dysfunctions those closely related to mitochondrial stress were also eveluated. Mitochondrial membrane potential was tested through cellular immunofluorescence and fluorescent method. After 24 h of stimulation with TG, decreased mitochondrial membrane potential (MMP), acting as the initial signal of mitochondrial stress, could be found through JC-10 dying (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Here, FCCP served as positive control during depolarization of plasma membrane potential.\u003c/p\u003e\n \u003cp\u003eSubsequently, proteins involved in mitochondrial fission (Fis1, DRP1/p-DRP1, MiD49 and MiD51) and fussion (OPA1, MFN1, MFN2) were determined during ER stress induction. The results verified that phosphorylated dynamin-related protein 1 (Drp1) on S616, mitochondrial fission 1 protein (Fis1) and mitochondrial dynamics proteins of 51 kDa (MiD51) were in down-regulation state, while mitochondrial dynamics proteins of 49 kDa (MiD49) were in increased expression. Mitochondrial fussion-related protein mitofusin-2 (MFN2) was declined. Crucially, optic atrophy\u0026minus;1 (OPA1) directly links mitochondrial structure and function. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, when the transmembrane potential across the inner membrane (\u0026Delta;\u0026Psi;m) is lost, long OPA1 isoforms (L-OPA1) is cleaved into short forms, which limited fusion and could facilitate mitochondrial fission. These data illustrated alterations in mitochondrial fission and fusion could also be detected during ER induction, implying that mitochondrial stress was also induced during TG stimulation.\u003c/p\u003e\n \u003cp\u003eFor further validation of the role of mitochondrial stress in the upregulation of ATF4, we transfected hUCMSCs with siRNA targeting OMA1 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, under ERS induction, simultaneous blockade of PERK and mitochondrial stress by GSK or siOMA1, respectively have advantage over blockade alone in ATF4 downexpression, which implied that ER stress and mitochondrial stress synergistically regulated ATF4 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur former research demonstrated that ERS hUCMSCs possessed better inhibition effect on RA Tfh cells by releasing PGE2 [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e], implying immune suppression of hUCMSCs could be enhanced by ER stress-induction. The present study demonstrated, for the first time, that ATF4 played a critical role in the enhancement of MSC-based therapy for RA through both ER and mitochondria stress. We observed that both PERK-eIF2\u0026alpha;-ATF4 signaling pathway from ER stress and OMA1-eIF2\u0026alpha;-ATF4 signaling pathway triggered by mitochondrial stress were involved in COX2 regulation.\u003c/p\u003e\n\u003cp\u003eCOX2, required for for the conversion of arachidonic acid into prostaglandins, such as PGE2, is upregulated in response to ER-stress induction and elevated COX2 will result in augmented PGE2 production, which acts as an inhibitor of T cell receptor signaling and thereby limits T cell activation [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. It was verified in reproductive research that ATF4 bound to COX2 promoter, and COX2-derived PGE2 can regulate ovulation [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. ATF4, a cellular stress induced-transcription factor, regulates a variety of genes involved in various physiological processes and plays a critical role as a stress-induced transcription factor. It orchestrates cellular responses, particularly in the management of ERS [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eER is the main organelle related to protein synthesis, modification, and processing in eukaryotic cells [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Mounting evidence suggests that altered ER homeostasis-events lead to the accumulation of unfolded or misfolded proteins in the ER lumen and breakdown of protein-folding homeostasis, creating a condition referred to ER stress [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. In response to ER stress, cytoprotective axes are triggered to restore protein homeostasis in the ER through unfolded protein response (UPR), which initiates pro-survival or pro-death responses and determines cell fate via the induction of PERK, ATF6 and IRE1 pathways[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBesides ER, mitochondria is another important organelle in cells. Mitochondria-associated endoplasmic reticulum membranes (MAMs) are dynamic coupling structures between mitochondria and ER, which combine the two critical organelle functions. So mitochondria and ER could regulate each other via MAMs [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our result manifested that ER stress could induce mitochondrial stress and eIF2\u0026alpha; phosphorylation is the core event of the endoplasmic reticulum-mitochondria coupling stress. eIF2\u0026alpha; phosphorylation induces the transcription factor ATF4. Moreover, mitochondrial stress is relayed to ATF4 through OMA1.\u003c/p\u003e\n\u003cp\u003eHerein, we further validated that during ER-stress triggering, the induction of COX2 was in an ATF4-dependent manner and elevated COX2 is associated with cellular stress response governed by the PERK-eIF2\u0026alpha;-ATF4 axis of the UPR pathway and OMA1-eIF2\u0026alpha;-ATF4 of the mitochondrial stress response. Our research confirmed that the ATF4 pathway activated by both ER-stress and mitochondrial stress stimulates COX2 transcription to enhance MSC-based therapy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTaken together, our data manifested that compared with unpre-treated hUCMSCs, ER-stressed hUCMSCs exhibited better immunosuppresive ability mainly through COX2 overexpression, which was regulated by ATF4. PERK-eIF2α activated by ERS and OMA1-eIF2α from mitochondrial stress co-regulate ATF4 expression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMSCs Mesenchymal stem/stromal cells\u003c/p\u003e\n\u003cp\u003eERS Endoplasmic reticulum stress\u003c/p\u003e\n\u003cp\u003eRA Rheumatoid arthritis\u003c/p\u003e\n\u003cp\u003eTfh T follicular helper cells\u003c/p\u003e\n\u003cp\u003eCOX2 Cyclooxygenase-2\u003c/p\u003e\n\u003cp\u003ePGE2 Prostaglandin E2\u003c/p\u003e\n\u003cp\u003eTG Thapsigargin\u003c/p\u003e\n\u003cp\u003eCIA Collagen-induced arthritis\u003c/p\u003e\n\u003cp\u003eTh1 T helper 1 \u003c/p\u003e\n\u003cp\u003eTh17 T helper 17\u003c/p\u003e\n\u003cp\u003eATF4 Activating transcription factor 4\u003c/p\u003e\n\u003cp\u003ePERK Protein kinase RNA like endoplasmic reticulum kinase\u003c/p\u003e\n\u003cp\u003ep-PERK Phosphorylated-PERK\u003c/p\u003e\n\u003cp\u003eHRI Heme-Regulated Inhibitor\u003c/p\u003e\n\u003cp\u003ePKR Protein Kinase R\u003c/p\u003e\n\u003cp\u003eGCN2 General Control Nonderepressible 2\u003c/p\u003e\n\u003cp\u003eeIF2\u0026alpha; Eukaryotic initiation factor 2\u0026alpha;\u003c/p\u003e\n\u003cp\u003ep-eIF2\u0026alpha; Phosphorylated-eIF2\u0026alpha;\u003c/p\u003e\n\u003cp\u003eER Endoplasmic reticulum\u003c/p\u003e\n\u003cp\u003eUPR Unfolded protein response\u003c/p\u003e\n\u003cp\u003eIRE1\u0026alpha; Inositol requiring enzyme 1\u003c/p\u003e\n\u003cp\u003eATF6 Activating transcription factor 6 \u003c/p\u003e\n\u003cp\u003ePGE2 Prostaglandin E2\u003c/p\u003e\n\u003cp\u003ehUCMSCs \u003cstrong\u003eHuman Umbilical Cord Mesenchymal Stem Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTF Transcription factor\u003c/p\u003e\n\u003cp\u003eMAM Mitochondria associated membranes\u003c/p\u003e\n\u003cp\u003eOMA1 Overlapping with m-AAA protease\u003c/p\u003e\n\u003cp\u003eMMP Mitochondrial membrane potential\u003c/p\u003e\n\u003cp\u003eMiD51 Mitochondrial dynamics proteins of 51\u0026thinsp;kDa\u003c/p\u003e\n\u003cp\u003eMiD49 Mitochondrial dynamics proteins of 49\u0026thinsp;kDa\u003c/p\u003e\n\u003cp\u003eMFN2 Mitochondrial fusion-related protein mitofusin-2\u003c/p\u003e\n\u003cp\u003eOPA1 Optic atrophy-1 \u003c/p\u003e\n\u003cp\u003eL-OPA1 Long OPA1 isoforms\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were in accordance with the guidelines of the Experimental Animals Management Committee (Liaoning Province, China), and were approved by the Experimental Animals Welfare \u0026amp; Ethical Committee (Date:16. 05. 2024, No. AEE24013), Dalian Medical University. Project name: Research on Pathogenesis and Treatment of Autoimmune Diseases. Human MSCs and RA peripheral blood samples were obtained from consenting volunteers enrolled in this study at the Second Hospital of Dalian Medical University. This study was approved by the Ethics Committee of the Second Hospital of Dalian Medical University (Date: 08. 10. 2023, ethical approval number: 2023-253). Project name: Pathogenesis Research and Immunotherapy in Patients with Autoimmune Diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll additional files are included in the manuscript. Using the OmicStudio tools, it is possible to predict the target genes regulated by ATF4 in both the TRRUST database and the Cistrome Data Browser database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJiaqing Liu: Formal analysis, Investigation, Writing \u0026ndash; review \u0026amp; editing. Xing Zhang: Formal analysis, Investigation, Methodology, Writing \u0026ndash; original draft. Xiangge Zhao: Methodology, Writing \u0026ndash; review \u0026amp; editing. Jinyi Ren: Formal analysis, Writing \u0026ndash; review \u0026amp; editing. Huina Huang and Cheng Zhang: Formal analysis, Investigation, Writing \u0026ndash; original draft. Xianmei Chen: Writing \u0026ndash; review \u0026amp; editing. Weiping Li: Human tissue providing. Jing Wei: Article designing, Writing \u0026ndash; drafting \u0026amp; revising. Xia Li: Project administration, Funding acquisition, Validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82071834, 82271839), Liaoning Undergraduate Program for Innovation and Entrepreneurship (S202310161043, S202310161019), Dalian key laboratory of human microorganism homeostasis and immunological mechanism research of diseases, Liaoning Provincial Education Department Basic Research Project (LJ212410161034, LJ212410161038) and Dalian Medical University Interdisciplinary Research Cooperation Project Team Funding (JCHZ2023010). The authors declare that they have not used artificial intelligence (AI)-generated work in this manuscript. The authors would like to acknowledge all lab members for insightful discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Y, Fang J, Liu B, Shao C, Shi Y. Reciprocal regulation of mesenchymal stem cells\u0026ensp;and\u0026ensp;immune\u0026ensp;responses.\u0026ensp;Cell Stem Cell. 2022; 29: 1515-30. \u003c/li\u003e\n\u003cli\u003eJiang B, Yao G, Tang X, Yang X, Feng X. MSCs relieve SLE by modulation of Th17 cells through MMPs\u0026ndash;CCL2\u0026ndash;CCR2\u0026ndash;IL-17 pathway. Rheumatol Autoimmun. 2021; 1: 30-9.\u003c/li\u003e\n\u003cli\u003eMaji S, Aliabouzar M, Quesada C, Chiravuri A, Macpherson A, Pinch A, et al. 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Mol Cell. 2018; 69: 169-81. \u003c/li\u003e\n\u003cli\u003eHetz C, Chevet E, Oakes SA. Proteostasis control by the unfolded protein response. Nat Cell Biol. 2015; 17: 829-38. \u003c/li\u003e\n\u003cli\u003eKaragoz GE, Acosta-Alvear D, Walter P. The Unfolded Protein Response: Detecting and Responding to Fluctuations in the Protein-Folding Capacity of the Endoplasmic Reticulum. Cold Spring Harb Perspect Biol. 2019; 11: a033886.\u003c/li\u003e\n\u003cli\u003eKasahara A, Scorrano L. Mitochondria: from cell death executioners to regulators of cell differentiation. Trends Cell Biol. 2014; 24: 761-70.\u0026ensp;\u003c/li\u003e\n\u003cli\u003eHe Q, Qu M, Shen T, Su J, Xu Y, Xu C, et al. Control of mitochondria-associated endoplasmic reticulum\u0026ensp;membranes\u0026ensp;by protein S-palmitoylation: Novel therapeutic targets for neurodegenerative diseases.\u0026ensp;Ageing Res Rev. 2023; 87: 101920. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MSCs, ATF4, rheumatoid arthritis, reticulum-mitochondria coupling stress","lastPublishedDoi":"10.21203/rs.3.rs-5620379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5620379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMesenchymal stem/stromal cell (MSCs) therapy represents a potential therapeutic tool to treat RA, but loss of secretory property post delivery restricted clinical application. It has been verified that endoplasmic reticulum stress (ERS)-MSCs exhibited better inhibition on rheumatoid arthritis (RA) T follicular helper cells (Tfh) via cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) activation with unknown molecular mechanism, particulary the overall outcome of ERS-modified MSCs on RA.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo compare the therapeutic efficacy, thapsigargin (TG)-stimulated or unstimulated MSCs were transplantated into collagen-induced arthritis (CIA) mice. Joint inflammation was evaluated from general and histological aspects. Splenocytes were isolated and flow cytometry was performed to assess the proportion of T helper 1 (Th1), Th17 and Tfh subsets. During mechanism exploration, TRRUST and Cistrome Data Browser databases were used to analyze transcription factors related to COX2 regulation, as well as target genes regulated by activating transcription factor 4 (ATF4). Then western blot and qRT-PCR were employed to determine the level of ATF4 in ERS-MSCs. To verify the function of ATF4 \u003cem\u003ein vivo\u003c/em\u003e, ATF4-overexpression MSCs were transplanted to CIA mice, joint inflammation, Th1, Th17 and Tfh subsets were analysed. To clear the molecular regulatory mechanism leading to ATF4 activation, protein levels of protein kinase RNA like endoplasmic reticulum kinase (PERK)/phosphorylated-PERK (p-PERK) and eukaryotic initiation factor 2α (eIF2α)/phosphorylated-eIF2α (p-eIF2α) were examined. Besides, ATF4 and eIF2α/p-eIF2α were checked after PERK blocking. Subsequently, mitochondrial stress was checked in ERS-MSCs. At last, blocking ERS and mitochondrial stress separately or simultaneously, ATF4 and eIF2α/p-eIF2α were checked again.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with MSCs, ERS-MSCs exhibited better therapeutic efficacy in CIA mice. Public databases and bioinformatics analysis confirmed the regulatory role of ATF4 on COX2 and experimental methods further confirmed ATF4-transfected MSCs diminished the joint inflammation of CIA mice. We also demonstrated that during ERS induction, PERK-mediated eIF2α phosphorylation contributes to elevated ATF4 expression. Besides, mitochondrial stress was also provoked in ERS-MSCs, coupling with ERS synergistically regulated ATF4.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eERS-MSCs exhibited better immunosuppresive ability than un-pretreated MSCs through COX2 overexpression, which was regulated by ATF4. Besides, ERS and mitochondrial stress co-regulate ATF4 expression. This study established a new role of ATF4 in promoting secretory properties of MSC and provided a promising MSC-based therapeutic strategy for RA treatment.\u003c/p\u003e","manuscriptTitle":"Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapy for rheumatoid arthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-23 16:54:59","doi":"10.21203/rs.3.rs-5620379/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-22T02:52:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-21T19:52:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-15T11:35:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-01-09T00:49:53+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-12-17T06:03:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c6f2c9d-249c-4a48-8322-364db540f3b0","owner":[],"postedDate":"January 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:09:06+00:00","versionOfRecord":{"articleIdentity":"rs-5620379","link":"https://doi.org/10.1186/s13287-025-04362-x","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-05-28 15:57:51","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2025-01-23 16:54:59","video":"","vorDoi":"10.1186/s13287-025-04362-x","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04362-x","workflowStages":[]},"version":"v1","identity":"rs-5620379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5620379","identity":"rs-5620379","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}