Rejuvenation of neutrophils and their extracellular vesicles is associated with enhanced aged fracture healing

Zhang, Xin; Baht, Gurpreet S.; Ron, Huang; Chen, Y-H.; Molitoris, Kristin Happ; Miller, Sara; Kraus, Virginia B.

doi:10.1111/acel.13651

Cited by 16 publications

(24 citation statements)

References 53 publications

(113 reference statements)

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“…It has been well-documented that EVs are involved in aging processes through regulating multiple biogenesis pathways, such as cellular senescence, inflammation, tissue regeneration, oxidative stress, metabolism and autophagy ( 1 , 7 , 19 , 40 , 41 ). We have observed age-associated declines of multiple immune cell-related EV subpopulations in healthy humans and mice, consistent with an immunosenescence phenotype in healthy aging that is reflected in EV subpopulations ( 1 , 6 ). Moreover, EV biomarkers indicating immunosenescence can be accentuated by common physical stresses in older adults, such as bone fracture and osteoarthritis ( 1 , 7 , 19 ).…”

Section: Discussionmentioning

confidence: 99%

Immune system-related plasma extracellular vesicles in healthy aging

Zhang,

Ma,

Huebner

et al. 2024

Front. Immunol.

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ObjectivesTo identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults.MethodsEV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers.ResultsCompared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1β, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1β, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation.ConclusionEndo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

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Section: Discussionmentioning

confidence: 99%

Immune system-related plasma extracellular vesicles in healthy aging

Zhang,

Ma,

Huebner

et al. 2024

Front. Immunol.

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“…In the context of miR, PBP has been shown to yield higher recovery of EV derived miRs ( Chung et al, 2020 ). As discussed by Zhang and colleagues ( Zhang et al, 2022 ) there is no established “gold standard” for EV isolation applicable to all subsequent analyses, therefore we chose PBP because of higher EV yields given our specific interest of profiling miRs from the myobundle and myobundle derived EVs. Additionally, miRNAseq is still a relatively new technique with numerous methods available for data processing.…”

Section: Discussionmentioning

confidence: 99%

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

et al. 2022

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Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle (“myobundles”). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

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“…Fractures were performed as previously described. 25–27 Briefly, mice were anesthetized using an induction chamber with 2% isoflurane and an oxygen flow rate of 1 L/minute. Sedation was ensured using paw-pinch test and the animal was placed on the surgical area.…”

Section: Methodsmentioning

confidence: 99%

The impact of age and sex on the inflammatory response during bone fracture healing

Molitoris,

Balu,

Huang

et al. 2024

JBMR Plus

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Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory “M1” and anti-inflammatory “M2” macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1β, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice – thus we termed these “age-accumulated” cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as “fracture response” cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: 1) age-accumulated cytokines, 2) female-specific age-accumulated cytokines, 3) and fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.

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Rejuvenation of neutrophils and their extracellular vesicles is associated with enhanced aged fracture healing

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 16 publications

References 53 publications

Immune system-related plasma extracellular vesicles in healthy aging

Immune system-related plasma extracellular vesicles in healthy aging

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

The impact of age and sex on the inflammatory response during bone fracture healing

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