Correction: Mitochondrial transplantation ameliorates experimental autoimmune encephalomyelitis by modulating the Th17/Treg balance and restoring metabolic homeostasis

Multiple sclerosis (MS) is a chronic autoimmune disease characterized by the immune system attacking the central nervous system (CNS), specifically targeting the myelin sheath that surrounds nerve fibers. This demyelination disrupts signal transmission, leading to neurological impairments such as sensory disturbances, motor dysfunction, and cognitive decline (1).Current therapeutic strategies for MS primarily focus on immunomodulation; however, they show limited efficacy in preventing neurodegeneration or promoting repair, particularly in progressive forms where pathology is dominated by axonal damage and mitochondrial dysfunction (2,3). Mitochondria are essential organelles responsible for producing ATP through oxidative phosphorylation (OXPHOS) and regulating calcium, apoptosis, and ROS generation (4,5). While mitochondrial impairment in MS lesions contributes to energy imbalance and neuronal apoptosis (6)(7)(8)(9). It is now increasingly recognized that mitochondrial fitness is also a fundamental determinant of immune cell fate and function. In the context of MS and EAE, the metabolic state of T cells and myeloid cells dictates their inflammatory potency. Specifically, pathogenic Th17 cells rely on aerobic glycolysis, whereas Treg cells favor mitochondrial OXPHOS to maintain immune tolerance (10). Mitochondrial dysfunction in these immune cells -characterized by impaired OXPHOS and excessive ROS-leads to the sustained activation of inflammatory pathways and a failure in immune tolerance (11)(12)(13)(14)(15). Additionally, the polarization of macrophages toward a pro-inflammatory M1-like phenotype is driven by a metabolic shift from OXPHOS to glycolysis, further exacerbating the inflammatory milieu (16). Therefore, mitochondrial transplantation has emerged as a promising strategy, potentially offering systemic benefits by not only providing preservation of tissue integrity but also by restoring metabolic homeostasis and reprogramming immune cell phenotypes.Drawing upon evidence from spinal cord injury and other CNS injury models-where transplanted mitochondria have been shown to be actively internalized by host cells such as macrophages and axons to restore bioenergetic function (17,18) exogenous mitochondrial delivery represents a novel approach to metabolically "rescue" compromised cells.Mechanistically, it has been established that the intercellular transport of mitochondria is a regulated process, often facilitated by Miro1-mediated movement through tunneling nanotubes, which enhances the rescue efficacy in stressed environments (19).Furthermore, studies have demonstrated that these extracellular mitochondria can successfully transit into host cells and integrate into the endogenous mitochondrial network to maintain functional integrity (20).Preclinical studies have demonstrated that introducing healthy mitochondria into damaged neural tissues can restore mitochondrial function, reduce inflammation, and improve cognitive and motor functions, in models of traumatic brain injury and ischemia (18,21), Parkinson's and Alzheimer's diseases (22,23), and status epilepticus (24). However, despite these advancements in local CNS injury models, the systemic impact of mitochondrial transplantation on the overall immune landscape of the EAE model remains unexplored.This study aims to explore the impact of mitochondrial transplantation in an EAE mouse model of MS, focusing on how restoring mitochondrial function through transplantation could modulate immune responses and inflammation, potentially altering disease outcomes. We examined how mitochondrial transplantation influences immune regulation and pathological changes in EAE. Specifically, we analyzed the proportions of key T-cell subsets and evaluated fibrotic markers in spinal cord tissues to elucidate the immunomodulatory and anti-fibrotic effects of mitochondrial therapy.Seven-week-old male C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). All animals were maintained under specific-pathogen-free (SPF) conditions with ad libitum access to water and a standard laboratory diet (Ralston Purina, St. Louis, MO, USA). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea (ID number: CUMC-2020-0048-02) and were performed in accordance with the ethical guidelines for animal experimentation.Mice were immunized via subcutaneous injection with 500 mg of MOG35-55 peptide emulsified in incomplete Freund's adjuvant (Chondrex, Redmond, WA, USA) supplemented with 500 mg of inactivated Mycobacterium tuberculosis (Difco, Franklin Lakes, NJ, USA). The emulsion was administered at two separate sites on the bilateral flanks. Additionally, 200 ng of pertussis toxin (Sigma, St. Louis, MO, USA) was administered Intraperitoneal Injection on days 0 and 2 to facilitate the induction of the disease. The mice were then observed and scored on a scale of 0-5 (with gradations at 0.5 intervals, allowing for intermediate scores): 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; and 5, death. The pathology scoring was conducted by two proficient technicians using a blind test. Each group contained five mice, and all experiments were repeated three or more times. All animals were euthanized using carbon dioxide (CO 2 ) inhalation in accordance with the AVMA Guidelines for the Euthanasia of Animals. CO 2 gas was introduced into the chamber at a controlled rate of 40% of the chamber volume per minute, following the displacement method recommended by the guidelines. Pre-filling of the chamber was avoided. Animals were monitored for complete cessation of respiration and heartbeat to confirm death.Mitochondria were isolated from L6 rat skeletal muscle cells using a Mitochondrial Isolation Kit for Cultured Cells (89874; Thermo, Waltham, MA, USA). In summary, 3×10 6 L6 cells were incubated with 400 mL of reagent A for 2 min on ice, with vortexing occurring every minute, and subsequently incubated with 5 mL of reagent B for 5 min on ice. Following the addition of 400 mL of reagent C, the cells were subjected to a centrifugation process at 700 × g for a duration of 10 min at a temperature of 4 °C. The resultant pellet was then transferred to a new tube and subjected to a further centrifugation at 12,000 × g for 15 min at 4 °C. The resulting pellet (mitochondria) was then washed with 400 mL of reagent C and subsequently subjected to a final centrifugation at 12,000 × g for 5 min at 4 °C. Protein concentrations were then quantified by bicinchoninic acid assay (23235; Thermo). In the EAE mouse model, mitochondria were administered intravenously via the tail vein once weekly at a dose of 10 mg in a total volume of 1 mL, whereas control mice received an equivalent volume of saline.Splenocytes were isolated from the spleens of the mice. The spleens were harvested and dissociated to obtain a single-cell suspension. To remove red blood cells, the cells were treated with ACK (Ammonium-Chloride-Potassium) lysing buffer, followed by centrifugation at 1,500 rpm for 5 minutes at 4 °C. The isolated splenocytes were re-suspended and cultured in RPMI 1640 medium supplemented with 5% bovine serum albumin (BSA).The study protocol was approved by the Institutional Review Board (IRB) of Seoul St. Mary's Hospital, The Catholic University of Korea (IRB number: KC24TISI0369), and written informed consent was obtained from all participants. All procedures were performed in accordance with the ethical standards of the Declaration of Helsinki. PBMCs were obtained from three female patients diagnosed with relapsing-remitting multiple sclerosis (RRMS). The patients were 20, 30, and 31 years old, with disease durations of 2.22, 7.19, and 12.52 years, respectively. All patients were receiving treatment with interferon beta-1a and were in a remission phase at the time of blood sampling. PBMCs were isolated from the peripheral blood of healthy controls (HCs) and patients with MS using Ficoll-Hypaque density gradient centrifugation (density 1.077 g/mL; Pharmacia Biotech, Uppsala, Sweden). Diluted blood was layered on Ficoll and centrifuged at 400 × g for 30 minutes (brake off). The mononuclear cell layer was harvested, washed twice with PBS, and treated with RBC lysis buffer to ensure purity. The isolated PBMCs were then resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for further experiments.After euthanasia, mice were transcardially perfused with cold PBS and fixed with 10% neutral buffered formalin. The spinal cords were then dissected and processed for paraffin embedding. Fivemicrometer-thick sections were prepared using a microtome (Leica Biosystems, Wetzlar, Germany). To evaluate pathological changes, sections were stained with Hematoxylin and Eosin (H&E) to assess inflammatory cell infiltration, and Luxol Fast Blue (LFB) with a hematoxylin counterstain to visualize the extent of demyelination. Pathological lesions were quantified using a semi-quantitative scoring system on a scale of 0 to 4. For inflammatory infiltration (H&E), the scores were defined as: 0, no infiltration; 1, small infiltration; 2, < 1/3 of white matter infiltrated; 3, > 1/3 of white matter infiltrated; and 4, severe inflammation. For demyelination (LFB), the scores were: 0, no demyelination; 1, mild demyelination; 2, < 1/3 of the demyelination area; 3, > 1/3 of the demyelination area; and 4, severe demyelination. All evaluations were performed by two independent observers in a blinded manner.To confirm the delivery of exogenous mitochondria and evaluate immune cell subsets, flow cytometry was performed using a FACS Calibur instrument (BD Biosciences, San Jose, CA, USA). To track mitochondrial transfer, L6 cells were pre-labeled with 200 nM MitoTracker Deep Red (MTDR; M22426, Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes at 37 °C before mitochondrial isolation, ensuring that only the exogenous mitochondria were tracked upon delivery to recipient cells. These pre-labeled mitochondria were then cocultured with human PBMCs to confirm their successful internalization into the recipient cells. To assess mitochondrial ROS levels, treated human PBMCs were incubated with 5 mM MitoSOX (M36008; Thermo Fisher Scientific) for 10 minutes at 37 °C. For the identification of T cell subsets, harvested cells were stained with species-specific fluorochrome-conjugated antibodies at a 1:100 dilution. For intracellular markers, including IL-17A and Foxp3, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Kit (eBioscience Waltham, MA, USA) according to the manufacturer's instructions. To identify human Th17 cells, PBMCs were stained with PE-Cy7-conjugated anti-CD4 (BioLegend, 300512) and PE-conjugated anti-IL-17A (eBioscience, 12-7179-42), while human Treg cells were identified using PE-Cy7conjugated anti-CD4, APC-conjugated anti-CD25 (BioLegend, 302610), and FITC-conjugated anti-Foxp3 (eBioscience, 11-4776-42). Similarly, to identify mouse Th17 cells, splenocytes were stained with PerCP-Cy5.5-conjugated anti-CD4 (eBioscience, 45-0042-82) and APC-conjugated anti-IL-17A (eBioscience, 17-7177-81), whereas mouse Treg cells were identified using PerCP-Cy5.5conjugated anti-CD4, APC-conjugated anti-CD25 (BioLegend, 102012), and PE-conjugated anti-Foxp3 (eBioscience, 12-5773-82). All flow cytometric data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).Serum levels of MOG-specific IgG2 were determined by indirect ELISA. 96-well plates were coated overnight at 4 °C with 10 mg/mL of MOG35-55 peptide in carbonate buffer. After blocking with 1% BSA in PBS for 1 hour, diluted serum samples were added to the wells and incubated for 2 hours at room temperature. The levels of MOG-specific antibodies were then detected using the IgG2 USA) according to the manufacturer's instructions. The was at using an San Jose, CA, spinal cord sections were and The sections were then incubated overnight at 4 °C with the following antibodies at a and and were incubated with then incubated with for 30 were using spinal cord sections stained with rat rat IL-17A and FITC-conjugated rat and rat mouse anti-Foxp3 were to the samples and to at 4 °C for The stained samples were then observed under a Germany). The of stained cells was conducted by two proficient technicians by a blind test. 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