Exercise facilitates post-stroke recovery through mitigation of neuronal hyperexcitability via interleukin-10 signaling

Abstract

Physical exercise is an effective therapy for improving stroke recovery. However, the exact underlying molecular mechanisms of exercise-enhanced neuronal repair remain unclear. As exercise affects the immune system in healthy individuals, and the immune system in turn influences recovery after stroke, we hypothesized that immune mechanisms play a role in exercise-induced neurological recovery. Using a model of ischemic stroke in adult male mice, we here show that the presence of regulatory T cells (Treg) within the ischemic brain is a prerequisite for exercise-enhanced functional and structural recovery. Treg prevent excessive and sustained hyperexcitability of periinfarct neurons via IL-10 signaling. This reduced hyperexcitability precedes alterations in neuronal connectivity, which underlie functional improvement. Together, we delineate the interaction of exercise-therapy, the immune system and functional recovery after ischemic stroke. Our findings can have translational relevance for further development of immune-targeted therapies.

Fig. 1. Exercise improves the functional and structural recovery after stroke in wild type mice.

a The adhesive tape removal test was used for functional outcome assessment. The removal index is calculated as follows: (removal time of impaired forelimb—removal time of unimpaired forelimb)/(removal time of impaired forelimb + removal time of unimpaired forelimb). b Exercise significantly reduced the asymmetry score in the adhesive tape removal test 49 days after photothrombotic stroke (*p = 0.04, two-sided t-test, n = 12 and 8 animals per group), which indicates improved functional outcome (ex = exercise, no ex = no exercise). c Mean infarct volumes were 2.64 mm³ ± 0.33 mm³ and 3.35 mm³ ± 0.36 mm³ (p = 0.16, two-sided t-test, n = 7 animals per group). d Exemplary coronary and sagittal MRI scans illustrate infarct size and location. e Axonal tracers CB and BDA were stereotactically injected into both motor cortices thus anterogradely labeling axons of pyramidal tract axons. f CB- and BDA-labeled axons were visualized immunohistochemically and crossing fibers were quantified at the level of the facial nucleus. g Running wheel training increased the number of crossing fibers, both ipsilaterally and contralaterally (**p = 0.003 and *p = 0.04, two-sided t-test, n = 9 and 12 animals per group), suggesting that exercise enhances axonal plasticity after stroke. h To better characterize exercise effects on neuronal network reorganization, we employed MRI-DTI fiber tracking (Color code: Fiber orientation: green: dorso-ventral, red: left-right, blue: rostro-caudal). i MRI-DTI fiber tracking demonstrated an increased number of interhemispheric connections after running wheel training (**p = 0.0043, two-sided t-test, n = 7 animals per group). j Exemplary scans of animals with a high (ex) and a low (no ex) number of interhemispheric connections. For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median, and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.

Fig. 2. Regulatory T cells (Tregs) are promotors of exercise-enhanced functional and structural recovery after stroke.

a RAG1^−/−-mice were subjected to photothrombotic stroke, received different subsets of T cells or vehicle (veh), and were then subjected to running wheel training. b Running wheel training did not influence the asymmetry index in the adhesive tape removal test (p = 0.46, two-sided t-test, n = 6 and 5 animals per group) in RAG1^−/−-mice without adoptive T cell-transfer (ex = exercise, no ex = no exercise). c Adoptive transfer of CD3⁺ T cells, in the absence of exercise, did not enhance recovery in RAG1⁻/⁻ mice (p = 0.28, two-sided t-test, n = 8 and 8 animals per group). d Following adoptive CD3⁺-T cells-transfer, exercise led to a significantly reduced asymmetry index (***p = 0.0002, two-sided t-test, n = 13 and 12 animals per group), indicating improved functional outcome. e There was a strong trend towards better functional outcome after CD4⁺- compared to CD8⁺-T cell-transfer (p = 0.07, two-sided t-test, n = 7 and 11 animals per group). f The transfer of FoxP3⁺ regulatory T cells (Tregs) resulted in significantly better functional outcomes than the transfer of FoxP3^- nonregulatory T cells (***p = 0.0009, two-sided t-test, n = 6 and 10 animals per group). g Preventing leukocytes from entering the brain by inhibition of very late antigen-4 (VLA-4) impaired stroke recovery (*p = 0.02, two-sided t-test, n = 7 animals per group; iso = isotype antibody). h MRI-DTI fiber tracking demonstrated an increased number of interhemispheric connections in mice that received Tregs before running wheel training compared to mice that received only vehicle and running wheel training (**p = 0.0013, two-sided t-test, n = 9 and 10 animals per group). Exemplary scans of animals with a high (ex) and a low (no ex) number of interhemispheric connections. For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 3. Exercise increases the proportion of Tregs in brain and cervical lymph nodes.

a Quantification of immune cells by flow cytometry 14 days after stroke revealed that exercise significantly reduced brain-infiltrating CD45^high immune cells and CD3⁺ T cells (*p = 0.04 and **p = 0.002, two-sided t-test, n = 7–9 animals per group), but had no impact on the count of CD45^high immune cells and CD3⁺ T cells in cervical lymph nodes (cLN) and spleen. b Exercise did not influence the proportions of CD4⁺- and CD8⁺-T cells in brain parenchyma, cervical lymph nodes and spleen 14 days after photothrombotic stroke (two-sided t-test, n = 9–12 animals per group). c Exercise increased the proportion of CD25⁺CD4⁺-and FoxP3⁺CD4⁺-regulatory T cells in brain parenchyma (*p = 0.04 and p = 0.1, two-sided t-test, n = 9–12 animals per group) and cervical lymph nodes (*p = 0.01, Mann-Whitney-test and **p = 0.01, two-sided t-test, n = 9–12 animals per group). d The expression of integrin CD49d, which facilitates leukocyte-trafficking into the brain, was upregulated in brain CD4⁺-T cells after exercise (*p = 0.03, Mann-Whitney-test, n = 9–12 animals per group), but not in CD4⁺-T cells isolated from cervical lymph nodes or spleen. The expression of the activation markers CD69 and CD11a on CD4⁺-T cells did not differ in any of the examined compartments. e Exercise led to a significant increase in the proliferation of FoxP3 + CD4+ regulatory T cells (Tregs) in the brain (*p = 0.04, two-sided t-test, n = 4–5 animals per group). Conversely, the proliferation of FoxP3-CD4⁺ and CD8⁺ effector T cells exhibited impairment both in the brain (*p = 0.03, two-sided t-test, n = 4–5 animals per group) and cervical lymph nodes (cLN) (*p = 0.03; **p = 0.006, two-sided t-test, n = 5–6 animals per group) as determined by Ki67 expression. For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.

Fig. 4. Exercise induces a shift towards anti-inflammatory immune cell polarization.

a Heatmaps based on immunohistochemical CD3-staining did not reveal apparent differences in the spatial distribution of immigrating CD3⁺-T cells between trained and untrained animals. Quantification of CD3⁺-T cells in different areas of interest in lesioned cortex, Thalamus and internal capsule 14 or 49 days after ischemia did not show differences in the cell count (two-sided t-test, n = 4–8 animals per group). b–d Representative images of immunohistochemical stainings from the infarct area and the internal capsule; similar results were observed in all animals analyzed (n = 28 across all groups). e To visualize immigrating FoxP3⁺-regulatory T cells in the brain parenchyma, we employed genetically modified FoxP3⁺-RFP-mice, in which FoxP3⁺-cells are labeled with the red fluorescent protein and can thus be detected by fluorescence microscopy. Heatmaps based on fluorescence microscopy did not reveal apparent exercise effects on the spatial distribution of immigrating FoxP3⁺-regulatory T cells in the brain parenchyma (n = 4 and 5 animals per group). f Volcano plot analysis of RT² profiler displays differential gene expression in mice with or without exercise 14 days after experimental stroke. Each data point represents a transcript, with the abscissa displaying log2 fold change and the ordinate statistical significance (negative log10 p value). Data are generated from 8 wildtype mice (no exercise n = 4; exercise n = 4, two-sided t-test). For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05. Source data are provided as a Source Data file.

Fig. 5. Exercise suppresses astrogliosis and enhances macrophage expression of markers associated with tissue repair.

a Astrocytes and microglia/macrophages were quantified in regions of interest cortex, Thalamus and internal capsule. b Representative immunohistochemical stainings of the glial fibrillary acidic protein (GFAP) and surface protein F4/40, performed to visualize astrocytes and microglia/macrophages; similar results were observed in all animals analyzed (n = 29 across all groups). c, d Exercise reduced astrogliosis in the lesioned cortex and the ipsilateral Thalamus 14 days post stroke (*p = 0.04 and p = 0.05, two-sided t-test, n = 4–11 animals per group). e, f Exercise led to decreased numbers of microglia/macrophages in the lesioned cortex and the contralateral Thalamus 14 days after stroke (*p = 0.02, two-sided t-test, and *p = 0.02, Mann-Whitney-test, n = 6-7 animals per group). In the chronic stroke phase after 49 days, exercise resulted in an increased cell count of microglia/macrophages in the lesioned cortex (*p = 0.04, two-sided t-test, n = 6–7 animals per group), whereas their numbers were decreased in the contralateral cortex (*p = 0.04, two-sided t-test, n = 6–7 animals per group). g, h Fourteen days after stroke, we found increased numbers of cells expressing Arginase-1, a marker commonly linked to repair-associated macrophage responses (*p = 0.02, two-sided t-test, n = 6–9 animals per group). Forty-nine days post-stroke, we observed a rise in the quantity of cells expressing CD206, indicative of a macrophage phenotype associated with tissue remodeling and homeostasis (*p = 0.02, t-test). For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05. Source data are provided as a Source Data file.

Fig. 6. Exercise modulates neuronal activity Treg-dependently.

a Graphic representation of a coronal slice and close-up of the periinfarct area indicating the recording site. Single-cell excitability was measured from a holding potential of -60 mV in response to a depolarizing current step (+160 pA, 2.5 s duration). b Fourteen days after stroke, neurons located in the periinfarct area of wild type mice showed an increased excitability as compared to sham-operated controls (*p = 0.05, Mann-Whitney test, n = 9 and 12 animals per group). c Exercise normalized stroke-induced hyperexcitability of periinfarct neurons in wild type mice (*p = 0.013, two-sided t-test, n = 11 and 12 animals per group). d Conversely, no exercise-mediated changes in AP generation were detected in sham animals (p = 0.3, Mann-Whitney test, n = 16 and 22 animals per group). e Similarly, exercise did not modulate the excitability of periinfarct neurons in RAG^-/- mice (p = 0.8, two-sided t-test, n = 4 animals per group). f Following Treg-transfer, exercise promoted a reduced excitability of periinfarct neurons in RAG^-/- mice, thereby suggesting that exercise modulates neuronal activity Treg-dependently (p = 0.07, two-sided t-test, n = 4 animals per group). For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05. Source data are provided as a Source Data file.

Fig. 7. IL-10 is a key mediator of exercise-induced stroke recovery.

a To exclude the possibility that the mere presence of activated CD4⁺ cells as a cofactor is sufficient to enable exercise-enhanced stroke recovery, we transferred either dysfunctional, FoxP3-deficient CD25⁺CD4⁺ T cells from scurfy mice or vehicle to RAG1^−/− mice. b The transfer of dysfunctional CD25⁺CD4⁺ T cells did not facilitate a better functional recovery compared to vehicle (p = 0.29, two-sided t-test, n = 8 and 6 animals per group), thus demonstrating that the mere presence of activated CD4⁺ T cells is not sufficient to enable exercise-induced stroke recovery. c The transfer of IL-10^−/−-Tregs to RAG1^−/− mice resulted in significantly worse functional recovery compared to the transfer of Tregs (**p = 0.003, two-sided t-test, n = 6 and 7 animals per group). d, e Similarly, MRI-DTI fiber tracking showed a lower number of interhemispheric connections in mice that received IL-10^−/−-Tregs compared to mice that received Tregs (*p = 0.03, two-sided t-test, n = 5 animals per group), thus emphasizing the key role of IL-10-secretion by Tregs for neuronal network reorganization. f, g To clarify, if IL-10 is also crucial for Treg-effects on neuronal activity, we transferred either IL-10^−/−-Tregs or Tregs to RAG1^−/−-mice and assessed exercise-effects on neuronal activity of periinfarct neurons. Our results show that Treg, but not IL-10^−/−-Treg, promoted exercise-mediated normalization of neuronal hyperexcitability, thus indicating that IL-10 secretion by Tregs has a pivotal role for exercise-induced neuronal repair (**p = 0.0013, two-sided t-test, n = 4 and 5 animals per group). For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: *p < 0.05, **p < 0.01 Source data are provided as a Source Data file.