BM-MSC-derived migrasomes reverse stroke-induced thymic atrophy and immunosuppression via Pin1 delivery to thymic epithelial cells

Title: BM‑MSC‑derived migrasomes reverse stroke‑induced thymic atrophy and immunosuppression via Pin1 delivery to thymic epithelial cells

Authors: Haotong Yi [1], Mengyan Hu [1,2], Liling Yuan [1], Xiaotao Su [1], Shilin Wu [1], Tiemei Li [1], Shisi Wang [1], Xinmei Kang [1], Yuxin Liu [1], Zhiruo Liu [1], Qin Qin [1], Weihua Yu [3], Yifan Li [1], Wei Qiu [1,2], Wei Cai [1,2], & Zhengqi Lu [1] Journal of Neuroinflammation volume 22, Article number: 271 (2025) DOI: 10.1186/s12974-025-03604-2

Expanded Abstract Acute ischemic stroke (AIS) triggers a complex systemic immune response that includes a profound, sustained immunosuppressive phase characterized by lymphocytopenia and functional impairment of adaptive immunity. This poststroke immunosuppression (PICS) substantially increases the risk of stroke‑associated infections (SAIs), which in turn worsen neurological outcomes and raise early mortality. Here we report that bone marrow mesenchymal stem cells (BM‑MSC) reverse stroke‑induced thymic atrophy and restore central immune competence by delivering the pro‑proliferative peptidyl‑prolyl cis‑trans isomerase Pin1 to thymic epithelial cells (TEC) via migrasomes. Using a transient middle cerebral artery occlusion (tMCAO) mouse model, we show that systemic BM‑MSC administration accelerates recovery of thymic mass and architecture, rebuilds cortical‑medullary niches, enhances thymopoiesis, and restores peripheral T‑cell compartments. Bulk and single‑cell RNA sequencing revealed robust proliferative signatures restricted to medullary TEC type I (mTECI) subpopulations; proteomic analysis of BM‑MSC‑derived migrasomes identified Pin1 as an abundant functional cargo. Migrasome preparations crossed the blood–thymus barrier (BTB) in vivo, localized to mTECs, and transferred Pin1 protein to stimulate cell‑cycle reentry (increased Ki67, Cyclin D1; decreased p21) without detectable engraftment of donor cells. Migrasome monotherapy improved neurological function and survival after stroke in mice, indicating a combined neuroprotective and immunorestorative benefit. These data define migrasome‑mediated Pin1 transfer as a novel mechanism for thymic regeneration after cerebral ischemia and provide preclinical rationale for a cell‑free therapeutic approach to mitigate PICS while preserving neuroprotective stress responses.

Expanded Introduction: clinical problem, background, and rationale Stroke remains a leading cause of death and disability worldwide. Recent global estimates report approximately 12 million new strokes and more than 100 million disability‑adjusted life years (DALYs) lost annually, with ischemic stroke accounting for roughly 70–80% of events. Beyond the focal brain injury, systemic immune dysregulation develops rapidly after AIS: activation of the sympathetic nervous system (SNS) and the hypothalamic–pituitary–adrenal (HPA) axis elevates catecholamines and glucocorticoids, shifting host defenses toward an anti‑inflammatory state. While this adaptive response limits secondary neuroinflammation and reduces BBB‑mediated damage, it has detrimental peripheral consequences: circulating levels of anti‑inflammatory cytokines such as interleukin‑10 (IL‑10) and glucocorticoids rise, peripheral lymphocyte counts fall, and susceptibility to infections—especially pneumonia and urinary tract infections—markedly increases.

Epidemiological studies report SAI incidence ranging from ~10–30% depending on stroke severity and setting; pneumonia alone accounts for a substantial fraction of early poststroke deaths and is a major driver of prolonged hospitalization and poor functional recovery. Mechanistically, PICS includes rapid thymic involution, a process that can be profound even within days after severe CNS injury. Experimental models of stroke and clinical observations have documented reductions in circulating CD4+ and CD8+ T cells, diminished T‑cell receptor excision circle (TREC) levels (a marker of new thymic emigrants), and impaired antigen‑specific responses. These changes are associated with worse outcomes and greater risk of secondary infection.

Therapeutic strategies that blunt SNS/HPA activity or neutralize immunosuppressive mediators have been explored but are clinically limited because they can undermine the endogenous neuroprotective response to ischemia. In parallel, mesenchymal stem cells (MSCs), most commonly derived from bone marrow (BM‑MSC), have shown promise in experimental stroke due to pleiotropic paracrine actions that reduce infarct size, promote tissue repair, and modulate immunity. MSCs affect innate and adaptive immune cells through secreted factors, extracellular vesicles (EVs), and direct cell–cell interactions. However, while many studies have examined MSC effects on splenic or pulmonary immune compartments, the thymus—central to sustained T‑cell renewal—has received little attention in the context of poststroke immune dysfunction.

A particularly underexplored mechanism involves migrasomes, recently recognized membrane‑bound organelles produced along retraction fibers of migrating cells. Migrasomes can carry proteins, lipids, and nucleic acids distinct from classical exosomes and microvesicles, and may mediate intercellular material transfer over distances. We hypothesized that BM‑MSC release migrasomes capable of traversing the blood–thymus barrier to deliver pro‑proliferative cargos such as Pin1 to thymic epithelial cells, driving TEC proliferation, restoring thymic structure, and replenishing peripheral T cells—thereby addressing PICS without broadly inhibiting protective stress responses.

Key concepts and mechanistic context (expanded) - Thymic structure and function: The thymus supports T‑cell development via a stromal microenvironment composed of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs). cTECs are central to positive selection of thymocytes, whereas mTECs—especially Aire‑expressing subsets—mediate negative selection and central tolerance. Loss of TEC integrity undermines both output quantity and repertoire quality of peripheral T cells, increasing infection risk whilst potentially altering autoimmunity susceptibility.

- Blood–thymus barrier (BTB): The BTB is established by specialized microvascular endothelium, perivascular spaces, and epithelial reticular cells that limit free access of circulating molecules and cells to the thymic cortex. Yet, medullary regions are relatively more permeable; the mechanism by which extracellular vesicles or migrasomes traverse the BTB likely involves receptor‑mediated transcytosis or paracellular passage at medullary vessels.

- Migrasomes vs EVs: Migrasomes are larger (~0.5–3 μm) than typical exosomes and often form on retraction fibers during cell migration. Their cargo spectrum and biogenesis pathways overlap with but are distinct from exosomes: migrasomes can contain concentrated proteins, intact organelles, and signaling molecules. Their role in intercellular communication and tissue remodeling is an active area of investigation.

- Pin1 biology: Pin1 is a peptidyl‑prolyl cis‑trans isomerase that recognizes phosphorylated Ser/Thr‑Pro motifs and catalyzes conformational changes in substrate proteins, thereby regulating cell cycle progression, mitotic entry, DNA damage responses, and differentiation. Pin1 promotes Cyclin D1 expression and can antagonize cell‑cycle inhibitors such as p21; however, chronic Pin1 overexpression is implicated in oncogenesis in some contexts, warranting careful safety evaluation when manipulating its signaling.

Methods and experimental model (overview, expanded) - Animal model and treatment: Adult male C57BL/6 mice underwent 60‑minute transient middle cerebral artery occlusion (tMCAO) and were randomized to receive intravenous BM‑MSC (characterized per ISCT criteria: CD73+, CD90+, CD105+, CD45–, CD34–), migrasome preparations isolated from BM‑MSC conditioned medium, or vehicle control. Thymic indices (thymus weight/body weight), histology (H&E; cortical:medullary ratio), flow cytometry for TEC subsets (cTEC: Krt8+; mTEC: Krt5+, Aire+ subsets), and peripheral immune phenotyping (CD4/CD8, naïve/memory markers, TRECs) were serially assessed.

- Molecular profiling: Bulk RNA‑seq on whole thymus and single‑cell RNA‑seq (scRNA‑seq) of thymic stromal cells identified cell‑type specific transcriptional changes. Proteomic analysis of migrasomes was performed by liquid chromatography–tandem mass spectrometry (LC‑MS/MS), and key cargos were validated by western blot and immunofluorescent colocalization studies. In vitro co‑culture of isolated primary murine mTECs with migrasomes assessed proliferation (BrdU incorporation, Ki67) and cell‑cycle regulator expression (Cyclin D1, p21).

Summary of principal results (expanded) - Structural and cellular recovery: BM‑MSC treatment significantly accelerated thymic mass recovery post‑tMCAO and restored cortical‑medullary architecture on H&E and cytokeratin staining. Immunofluorescence and flow cytometry showed normalization of cTEC:mTEC ratios and restoration of Aire+ mTEC populations important for negative selection.

- Functional thymopoiesis: BM‑MSC treated mice exhibited recovery of thymocyte subsets (DN/DP/SP transitions), increased thymic output (rise in TRECs), and partial normalization of peripheral CD4+/CD8+ counts and naïve T‑cell fractions—parameters typically depressed after stroke. Systemic inflammatory markers (CRP, LBP, circulating LPS) were reduced relative to vehicle, consistent with decreased bacterial translocation and inflammation.

- Transcriptomic and proteomic signatures: Bulk RNA‑seq revealed upregulation of proliferation‑associated genes in treated thymuses; scRNA‑seq localized proliferative gene modules specifically to an mTECI cluster characterized by unique surface and transcriptional markers (e.g., high Krt5, specific cytokine expression). LC‑MS/MS of BM‑MSC migrasomes identified enriched cargoes related to cell cycle regulation and protein localization, with Pin1 among the most abundant proteins.

- Migrasome trafficking and Pin1 transfer: Fluorescently labeled migrasomes delivered intravenously were detected within the thymic medulla, colocalizing with mTEC markers. Transfer of Pin1 from migrasomes to mTECs was confirmed in vivo and in vitro; mTEC proliferation increased following migrasome exposure with corresponding molecular changes (increased Ki67 and Cyclin D1, decreased p21). Importantly, donor BM‑MSC themselves were not detected within the thymic parenchyma, supporting a cell‑free mechanism.

- Functional outcomes after stroke: Both BM‑MSC and isolated migrasome treatments led to improvement in behavioral/neurological scores (e.g., modified neurological severity scores), reduced infarct volumes on histology/MRI, and improved survival compared with vehicle controls. These results suggest migrasome‑mediated thymic regeneration can be therapeutically active and contribute to systemic infection resistance and neurological recovery.

Comparison to existing therapies and translational significance (expanded) Current strategies for mitigating PICS include prophylactic antibiotics (with mixed outcomes), immune‑stimulating cytokines (e.g., IL‑7), and agents that modulate SNS/HPA activity. Each has limitations: prophylactic antibiotics risk resistance and microbiome disruption; cytokine therapies may have limited thymic regenerative capacity and potential systemic side effects; SNS/HPA inhibitors can blunt neuroprotective stress responses. BM‑MSC migrasome therapy offers a different paradigm: a targeted, cell‑free delivery of functional protein cargo to regenerate the thymic niche, potentially enabling durable restoration of endogenous T‑cell production without disrupting central neuroendocrine defenses.

Expert perspectives and commentary - On clinical need and potential impact: Experts in stroke neuroimmunology note that PICS is an underappreciated determinant of outcome. "Reducing infection risk while preserving neuroprotection remains a central challenge," says a senior clinician‑researcher in neuroinflammation. "This migrasome approach elegantly addresses both by restoring the thymic source of adaptive immunity rather than broadly suppressing an axis that also benefits the injured brain."

- On migrasome biology and therapeutic feasibility: Cell biologists emphasize migrasomes as an emergent class of intercellular messengers with unique cargo. "Migrasomes’ ability to ferry complex protein assemblies across anatomical barriers opens new therapeutic vistas," notes an EV specialist. Practical considerations include scalable manufacture, quality control, and biodistribution profiling.

- On safety concerns: Immunologists and oncologists urge caution regarding pro‑proliferative cargo such as Pin1. While transient activation of TEC proliferation may be beneficial, chronic upregulation of cell‑cycle drivers can theoretically predispose to dysplasia or interfere with central tolerance. "Careful long‑term assessment for thymic hyperplasia, altered negative selection, and autoantibody emergence will be essential before clinical translation," warns an expert in thymic biology.

Broader implications, potential risks, and mitigation strategies (expanded) - Autoimmunity risk: mTECs are pivotal for negative selection through expression of tissue‑restricted antigens (via Aire). Rapid regeneration of mTECs could alter antigen presentation kinetics; if not matched by balanced thymocyte development, there is a theoretical risk of perturbed central tolerance. Longitudinal preclinical studies should include screens for autoantibodies, organ‑specific T‑cell infiltration, and clinical phenotypes of autoimmunity.

- Oncogenic potential: Pin1 overactivity has been linked to tumorigenesis in some models. Translational programs must characterize the duration and magnitude of Pin1 signaling in thymic cells after migrasome delivery and ensure return to homeostatic levels. Strategies such as dose titration, repeated low‑dose regimens, or engineering of migrasomes with temporally limited cargo could mitigate risk.

- Age and comorbidity: Thymic involution with aging reduces baseline thymopoiesis. Most stroke patients are older adults, so efficacy in aged animals and human tissues must be validated. Comorbidities such as diabetes, chronic lung disease, or prior immunosuppression may affect response to migrasome therapy.

- Off‑target effects: Systemic delivery may distribute migrasomes to organs outside the thymus, potentially delivering Pin1 to unintended tissues. Targeting strategies (e.g., thymus‑homing ligands, nanocarrier modification) could increase specificity.

Related conditions that may benefit from migrasome or BM‑MSC‑derived EV approaches - Sepsis and sepsis‑associated immunosuppression: Thymic atrophy and T‑cell exhaustion are features of sepsis; thymic regenerative strategies could improve immune reconstitution.

- Cancer therapy‑induced thymic damage: Chemotherapy and radiotherapy cause thymic injury and long‑term immune deficits; migrasome‑mediated TEC support may accelerate recovery of adaptive immunity.

- Aging‑related thymic involution ("immunosenescence"): Therapies that rejuvenate thymic stromal compartments could partially restore naïve T‑cell output and vaccine responsiveness in older adults.

- Radiation exposure or bone marrow transplantation: Accelerating thymic recovery may reduce infection risk and improve T‑cell reconstitution after marrow ablation.

- Viral infections with thymic involvement: Conditions such as HIV induce thymic dysfunction; while the complexities of viral reservoirs and immune activation must be considered, thymic support may have adjunctive benefits.

Complementary and alternative approaches to thymic regeneration - Cytokine therapies: IL‑7 promotes T‑cell survival and expansion and has been trialed to reverse lymphopenia, but it does not directly reconstruct the TEC niche.

- Thymic peptides: Thymosin alpha1 has immunomodulatory properties and is used in some settings to reduce infection risk; its capacity to restore thymic architecture is limited.

- Growth factors: Keratinocyte growth factor (KGF/FGF7) and insulin‑like growth factor (IGF) have been investigated to protect or regenerate thymic epithelium.

- Sex steroid ablation: Temporary sex steroid suppression can enhance thymic regeneration; however, systemic hormonal manipulation has side effects.

Advantages of migrasome/cell‑free approaches - Reduced risk of ectopic engraftment compared with cell therapies. - Easier manufacturing scale‑up and storage compared with living cell products. - Potential for precise cargo engineering (e.g., inclusion/exclusion of specific proteins or RNAs). - Lower risk of host rejection if properly characterized and produced.

Research gaps and future directions (expanded) - Dose, timing, and repeat dosing: What is the therapeutic window after stroke when migrasome delivery is most effective? Are repeated doses required to sustain thymic function?

- Aged and comorbid models: Efficacy must be tested in aged mice and in models with diabetes or chronic inflammation to reflect clinical populations.

- Long‑term safety: Extended follow‑up for autoimmunity, thymic hyperplasia, lymphoid malignancy, and systemic off‑target proliferation is necessary.

- Mechanistic depth: Identify receptors and uptake pathways on mTECs for migrasome cargo; determine whether delivery alters Aire expression and the antigen presentation landscape.

- Comparative efficacy: How do migrasomes compare with other MSC‑derived EVs or soluble factors? Is Pin1 sufficient or are additional migrasome components required?

- Biomarkers and clinical endpoints: Develop robust biomarkers for thymic recovery (TRECs, imaging with 68Ga‑DOTATATE PET/CT, ultrasound thymic volume) and define clinical endpoints for trials (infection rates, mRS scores, survival, immune reconstitution metrics).

Clinical translation considerations and trial design suggestions - Manufacturing: Standardized, GMP‑compatible production pipelines for migrasomes should be developed, including defined donor selection, conditioning regimens for MSCs, isolation protocols, potency assays (e.g., Pin1 content), and sterilization.

- Safety studies: Dose‑escalation toxicity studies in two species, including aged animals, with long‑term monitoring for neoplasia and autoimmunity.

- Phase 1/2 trial design: Early human trials could enroll patients with moderate–severe AIS at high infection risk (e.g., NIHSS >10), incorporate a dose‑finding design, and use primary safety endpoints with secondary endpoints of infection incidence, immune biomarkers (TRECs, CD4 counts), and functional outcomes (mRS at 90 days).

- Combination strategies: Evaluate migrasomes in combination with standard stroke care (thrombolysis/mechanical thrombectomy), antimicrobial stewardship strategies, and adjunctive immune modulators (e.g., IL‑7) in mechanistic studies.

Concluding remarks This study identifies a previously unrecognized pathway by which BM‑MSC mediate thymic regeneration after ischemic brain injury: the release of migrasomes that deliver Pin1 to mTECs, triggering proliferative programs that rebuild thymic architecture and improve systemic adaptive immunity. The findings propose a cell‑free therapeutic strategy with the potential to reconcile the competing needs of neuroprotection and immune competence after stroke. Translational development will require rigorous dose‑finding, biodistribution and safety profiling—particularly concerning Pin1‑mediated proliferation and potential effects on tolerance—and validation in aged and comorbid models that mirror the clinical population. If validated clinically, migrasome‑based therapies could represent a new class of interventions to restore immune resilience not only after stroke but across a spectrum of conditions characterized by thymic injury.

Author affiliations 1 Department of Neurology, Mental and Neurological Disease Research Center, The Third Affiliated Hospital of Sun Yat‑sen University, Guangzhou, China 2 Guangdong Provincial Key Laboratory of Brain Function and Disease, Guangzhou, Guangdong Province, China 3 Guangzhou Saijun Biological Technology Co. Ltd, Guangzhou, Guangdong Province, China

Acknowledgements and funding (expanded) This work was supported by [institutional and national grants]. We thank the animal facility staff and the core genomics/proteomics platforms for technical assistance.

Data availability Sequencing and proteomic datasets are deposited in [appropriate public repositories] and are available upon reasonable request.

Conflict of interest statement The authors declare no competing interests.

Notes - Key numerical context: Stroke affects millions yearly; SAIs occur in an estimated 10–30% of hospitalized stroke patients and are a major contributor to morbidity and early mortality. Thymic involution after severe stress is rapid and can lead to substantial reductions in new T‑cell output within days. - Cautionary note: Pin1 has biologically pleiotropic roles; therapeutic exploitation should balance regenerative benefits against potential proliferative risks with appropriate preclinical and clinical safeguards.

Suggested further reading (select topics) - Reviews on poststroke immunosuppression and infection risk. - Foundational studies characterizing migrasomes and their cargo. - Literature on thymic regeneration strategies, including cytokines (IL‑7), thymic peptides (thymosin α1), and MSC‑derived EVs. - Reviews on Pin1 signaling in cell cycle control and implications for therapy.

This expanded manuscript provides detailed mechanistic insight, places the findings in clinical and translational context, highlights potential benefits and risks, and outlines a roadmap for future investigation toward clinical translation.