Young extracellular vesicles restore burn-induced adipose tissue immunometabolic and mitochondrial function in older mice

Aijaz, Ayesha; Bieerkehazhi, Shayahati; Kang, Navneet; de Brito Monteiro, Lauar; Saladhna, Sean; Faour, Sara; Challagundla, Bhavishya; Rix, Graham; Chogan, Faraz; Jeschke, Marc G.

doi:10.1038/s41598-025-19239-5

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Published: 09 October 2025

Young extracellular vesicles restore burn-induced adipose tissue immunometabolic and mitochondrial function in older mice

Ayesha Aijaz^1,2,
Shayahati Bieerkehazhi^1,2,
Navneet Kang⁴,
Lauar de Brito Monteiro^1,2,
Sean Saladhna²,
Sara Faour³,
Bhavishya Challagundla²,
Graham Rix²,
Faraz Chogan³ &
…
Marc G. Jeschke^1,2,3

Scientific Reports volume 15, Article number: 35328 (2025) Cite this article

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Abstract

The idea of blood as an elixir of youth led to the classical experiment of heterochronic parabiosis, which demonstrated that young blood could rejuvenate aged tissues in mice. Later, it was discovered that this rejuvenating capacity is due to the presence of circulating extracellular vesicles (EVs), which override deleterious signals from the aged environment via intercellular cues that promote tissue renewal. Aging is associated with alterations in the structure and cargo of EVs with diminished nucleic acid content. We know that aging is a chronological process of progressive cellular and tissue dysfunction that impairs the capacity of older trauma patients to adequately respond to stress, particularly burn trauma, with the adipose tissue serving as the central mediator. Our results demonstrated that, in addition to increasing senescence with chronological aging, burn injury further intensifies the senescence burden in adipose tissue. Notably, EVs from young mouse serum samples significantly reduced burn-induced senescence in aged adipose tissue. We further demonstrated that EVs mitigate lipolysis and are crucial in hepatocellular signaling for the regulation of hepatic inflammation, fat accumulation and dysfunction. Finally, we provide evidence that EV therapy reestablishes immuno-metabolic function, mitochondrial bioenergetics, immune cell infiltration and adipose tissue function.

Introduction

Older adults are the fastest-growing demographic population, and by 2050, they will represent almost 39% of trauma admissions across North America¹. Among trauma patients, severe burns are responsible for an estimated 300,000 deaths annually worldwide. Unfortunately, older burn patients have a substantially greater mortality rate of 75% compared to 15% in the young adults with an associated longer length of hospital stay^2,3. Survival and outcomes have improved significantly for pediatric and adult patients; however, regrettably, this remains a challenge in older burn patients. This is because the etiology behind the age-dependent pathophysiologic response to burns remains poorly understood. While systemic responses to burn injuries initially manifest as single-organ malfunctions², deleterious processes interact with extraordinary complexity within and between organs to rapidly drive the fatality of this condition². Aging is a chronological process of progressive cellular and tissue dysfunction that impairs the capacity of older trauma patients to adequately respond to stress. While the nine hallmarks or common denominators of aging⁴ have been clearly delineated, the challenge is to dissect the interconnectedness between the hallmarks and their relative contributions to aging and poor outcomes in the older adult population. Thus, the goal of modern medicine in the past few years has been to identify targets to improve human health during aging.

Surprisingly, scRNA sequencing of 17 organs demonstrated that age-associated changes in gene expression arise in subcutaneous and inguinal adipose tissue (AT) prior to changes in other organs⁵. In addition to changes in gene expression and the redistribution of AT to the visceral region, aging induces several structural and functional alterations within the AT, which impair the capacity of older trauma patients to adequately respond to stress. With age, the AT harbors a depot of senescent cells⁶ and accumulate a distinctive phenotype, referred to as the senescence-associated secretory phenotype (SASP), which inhibits self-renewal of adipose stem cells and differentiation into adipocytes and leads to a dysfunctional immune cell population within the stromal vascular fraction (SVF) of the AT. We have extensively studied the pathophysiological changes in the AT in burn patients. We discovered that while young adults respond to burn trauma by initiating a hypermetabolic response, older adults exhibit a hypometabolic response. Furthermore, in young adults, burn injury leads to increased lipolysis, browning, inflammation, increased systemic FFA influx, and adverse adipokine secretion; all of these responses are blunted in older adult burn patients⁷, leading to reduced survival. To this end, a fundamental process of chronological aging is the dysregulation of energy homeostasis, with the AT serving as the central metabolic organ in regulating whole-body energy homeostasis.

To improve outcomes in older adult burn patients, it is imperative to understand and target the age-imposed biochemical changes in the tissue niches that inhibit the performance of regenerative cell pools and lead to a decrease in tissue maintenance and repair and therefore a response to injury. Approximately one decade ago, the idea of blood as an elixir of youth led to the classical experiment of heterochronic parabiosis, which demonstrated that young blood could rejuvenate aged tissues in mice. Later, it was discovered that this rejuvenating capacity is due to the presence of circulating extracellular vesicles (EVs), which override deleterious signals from the aged environment via intercellular cues that promote tissue renewal. Aging is associated with alterations in the structure and cargo of EVs with diminished nucleic acid content. In contrast, EVs from young mouse serum have beneficial effects by restoring tissue function. Recently, the beneficial effects of young serum on the preservation of muscle cell mitochondrial bioenergetics and skeletal muscle regeneration were attributed to circulating EVs present in young serum. EVs have been shown to restore cellular bioenergetics and attenuate inflammaging by rejuvenating T-cell immunotolerance⁸; decreasing senescence markers and ROS levels; decreasing lipid peroxidation; and increasing the brown interscapular AT mass⁹. Furthermore, EVs have been shown to decrease the levels of cellular biomarkers of senescence in the liver, BAT, kidney, and serum⁹.

To this end, herein, we present a study in which we administered EVs from young mice sera in chronologically aged mice and following a burn injury. We present evidence that EV administration from young serum reduce senescence and improve AT function and mitochondrial bioenergetics in aged mice post-burn. We further demonstrate that EVs reestablish immune cell infiltration, restore immuno-metabolic function and attenuate cellular senescence in aged individuals (Fig. 1).

Results

Transmission electron microscopy

The EVs were derived from mouse serum and subjected to transmission electron microscopy (TEM) analysis. As depicted in Fig. 2A, the exosomes exhibited a characteristic cup-shaped or circular morphology with a size distribution ranging from 150 to 200 nm. Of note, the dark regions evident in the TEM image are attributed to the presence of uranium, a heavy metal that appears dark under TEM, in the staining agent.

EV MiRNA cargo

To investigate age-associated changes in serum EV cargo that may contribute to changes in function, we analyzed the expression of 380 miRNAs in older and adult mouse EV fractions. Overall, 174 miRNAs were detectable above the threshold (Supplementary Fig. 1). MiRNAs play important roles in senescence, mitochondrial biogenesis and function, adipogenesis and immune cell function. We focused on understanding the differential expression of AT and immunological miRNAs that regulate key immunological processes, including development, lineage commitment, effector functions and the aging of various innate and adaptive immune cells^10,11,12. Among these we observed differential expression in the most prominent clusters, including the miR-146/miR-155 axis; the miR-17-92 cluster, which consists of miR-17, miR-18a, miR-19a/b, miR-20a and miR-92; the miR-23∼27∼24 cluster; and the miR-223 and miR-181 (Fig. 2B–D).

We observed differential expression of the miR17-92 cluster, which has been implicated in age-related conditions. In particular, miR-17, miR-19a/b, and miR-92a/b were downregulated, whilst miR-18a and miR-20a were upregulated in aged mice EVs compared with young mice EVs (Fig. 2B). The miR 17–92 cluster directly targets both Smad2/4 and TGF-β signaling via TGF-β receptor II (TGFBRII)^13,14,15. Furthermore, a decrease in the expression of these miRNAs correlates with increased transcript levels of p21/CDKN1A indicating a direct implication in senescence. Next, we noted downregulation of miR-23a/b and miR-27b and the upregulation of miR-24 and miR-27a (Fig. 2C). The miR-23∼27∼24 cluster directly targets lymphoid transcription factors to regulate lymphoid cell differentiation and has been shown to promote myeloid lineage commitment and cell proliferation^{16,17,18,19,20,21}. Interestingly, miR-146a was significantly upregulated in aged EVs compared with young EVs (Fig. 2D). The roles of miR-146 and miR-155 in regulating inflammation are particularly important. miR-146a is an anti-inflammatory agent that negatively regulates TNFα- and IL-6-driven immune signaling to suppress differentiation processes and effector functions in innate immune cells^22,23,24,25. It also negatively regulates Th1 cell differentiation²⁶. Along with miR-146, the proinflammatory miRNA miR-155 positively regulates IFNγ production in CD4⁺ and CD8⁺ T cells^27,28 and is implicated in the complex spatiotemporal regulation of macrophages²⁹. In contrast, miR-223 reduces the induction of both proinflammatory and immunomodulatory genes in macrophages. To this end, we observed significant increases in the serum expression of miR‐223 in EVs from aged mice (Fig. 2D).

To understand the functional impact of these changes in miRNA expression, we assessed the predicted molecular functions and pathways affected by the differentially expressed miRNAs. Pathways vital for adipose function and immune signaling, such as pathways involved in adipocytokine signaling, liver disease, ER processes, HIF-1 and AMPK signaling, T-cell receptor signaling, and T-cell differentiation, were found to be enriched in the differentially expressed miRNAs (Fig. 2E,F).

Together, these data suggest that aging induces significant changes in the miRNA content of serum EVs which may account for the reduced effectiveness of EVs derived from aged subjects in countering the burn-induced pathophysiological processes.

Impact of burn on body weight, food intake, and adipose tissue postinjury

We administered EVs from young mice sera in chronologically aged mice following a burn injury (Fig. 3A). We assessed the impact of burn injury on mice food intake, body, and AT weights in aged and young mice and determined whether EV administration could affect these parameters. Interestingly, aged burn mice consumed significantly more food than young burn and aged sham mice. EV administration slightly increased food intake in aged sham mice but on the contrary decreased food consumption in aged mice after the burn (Fig. 3B). On the other hand, burn injury resulted in a loss of body mass in both aged and young mice, with a more severe loss in young mice while aged sham mice gained body mass after EV administration (Fig. 3C). Next, we sought to examine if these metabolic changes will correlate to AT weight changes as well. Strikingly, we observed that burn injury did not result in a change in inguinal white AT (iWAT) mass in young mice (Fig. 3D). Aged burn mice demonstrated a significant increase in iWAT mass compared to aged sham and young burn mice. Likewise, the epididymal WAT (eWAT) mass was also significantly greater in aged burn mice than in young burn mice (Fig. 3E). These data show that AT in aged mice is unable to respond to burn-induced metabolic changes. Meanwhile, with EV administration, AT exhibits less increase in its mass indicating that it starts to restore its function and normal responses.

Aging is associated with chronic senescence of adipose tissue, which is further aggravated by burn injury

We next sought to assess whether burn injury exacerbates chronic senescence in the ATs of aged mice. We measured the gene expression levels of p21 and p16, two well-established senescence markers (Fig. 4A,B). Specifically, p21 is a cyclin-dependent kinase inhibitor (CDKI) of the G1/S phase of the cell cycle and has antiproliferative functions that are p53-dependent. While chronological aging resulted in a 1.4-fold increase in p21 expression in aged sham mice compared to young sham mice, burn injury aggravated senescence, resulting in a 3.8-fold increase in p21 levels in aged burn mice. These findings revealed significant increases in senescence compared to aged sham and young burn. Remarkably, EV administration significantly reduced the p21 level in the AT of aged burn mice (Fig. 4A). Similar results were observed for p16, another CDKI that arrests the cell cycle in the G1 phase. P16 levels were 2-fold and 3.1-fold greater in the ATs of aged sham and aged burn mice, respectively, compared to young shams. These increases were significant compared to those in young burn ATs, which represented a 1.2-fold increase in p16 levels compared to those in young sham Ats (Fig. 4B). Thus, our results demonstrate that senescence increases with chronological aging, and burn injury further intensifies the senescence burden in the AT. Notably, EVs from young mouse serum samples significantly reduced burn-induced senescence in aged ATs.

EV administration post-burn may restore adipose tissue Browning in aged mice

We have previously shown that the adipose browning response to burn trauma is impaired with age³⁰. Here, we wanted to assess whether EV administration from young serum can restore browning in the AT following burn trauma. In accordance with the findings of previous work, aged ATs demonstrated significantly blunted expression of UCP-1, a key browning marker, compared to that of their younger counterparts (Fig. 4C,D). Aged burn ATs exhibited a slight increase in UCP-1 expression following EV administration. These findings unravel new opportunities to explore that EV administration may be a beneficial therapeutic strategy for reducing burn-induced hypermetabolism and AT browning in young adults and can conversely restore the AT browning response in aged counterparts.

To further understand the phenomena of burn-induced adipogenesis and mitochondrial biogenesis, we examined the expression of peroxisome proliferator-activated receptor γ (PPAR-γ), which is highly expressed in adipocytes. PPARγ is a ligand-dependent transcription factor that serves as a master regulator of adipogenesis, is central to thermogenesis and is an active modulator of lipid metabolism and insulin sensitivity. In addition, it has roles in cellular proliferation, inflammation, and immunoregulation. We observed a significant increase in PPARγ expression in young ATs in response to burn injury. In contrast, aged ATs did not respond with similar increase in PPARγ expression (Fig. 4E). Additionally, the expression of PGC1α, also known as peroxisome proliferator-activated receptor γ coactivator 1α (PPARγ), the transcriptional coactivator of PPARγ and the chief regulator of mitochondrial biogenesis and mitochondrial respiration, increased in the AT of young mice while aged mice failed to respond to burn injury with a concomitant increase in PGC1α expression (Fig. 4F). Interestingly, adiponectin expression levels responded significantly to EV administration. Adiponectn, an adipocyte secreted hormone which has an inhibitory effect on Ucp1 expression, showed an elevated expression in aged sham mice which was significantly increased after-burn. Nonetheless, EV administration significantly reduced adiponectin expression in the aged burn group (Fig. 4G). Notably, adiponectin level in the young burn group was significantly lower than their counterpart.

Taken together, these findings are consistent with our previous findings, further confirming an impaired browning response in aged mice. Likewise in response to EV administration, PGC1α expression was significantly lower in aged burn mice than in young mice.

EVs restore AT mitochondrial bioenergetics in aged mice postburn

A distinct feature of burn-induced hypermetabolism is increased mitochondrial activity in the AT and concomitant increased resting energy expenditure, particularly in young adults^31,32,33. To test this phenomenon, we assessed alterations in mitochondrial activity with age and postburn trauma. We further studied the impact of EV administration on mitochondrial bioenergetics by analyzing the oxygen consumption rate (OCR) in response to mitochondrial stress tests. As expected, the OCR were increased in AT of young mice following burn injury compared to shams (Fig. 5A). In contrast, OCRs were reduced throughout the assay in aged ATs, and burn trauma further promoted this reduction in OCRs in these aged mice. Interestingly, EV administration restored mitochondrial bioenergetics in aged sham and aged burn ATs, as demonstrated by an increase in the OCR (Fig. 5A). Further analysis revealed a significant reduction in basal respiration in aged burn ATs compared to young burn ATs and aged sham ATs, a result indicating impairment in mitochondrial activity due to both chronological aging and burn injury. Basal respiration improved significantly in aged burn ATs after EV administration (Fig. 5B). Similar results were observed for mitochondrial-dependent ATP production (Fig. 5C). Similarly, maximal respiration was impaired with age and burn injury (Fig. 5D), while it was significantly improved in both aged sham and aged burn AT rats following EV administration.

Aging is associated with the dysregulation of adipose immune cell population

Adipose tissue is particularly dynamic and responds to changes in the microenvironment. Both innate and adaptive immune cells coordinate immune surveillance responses to ensure AT homeostasis, and it is important to understand the changes in immune cell populations and their associated phenotypes both during chronological aging and in response to pathophysiological changes following burn injury. We observed that aging is associated with the dysregulation of adipose tissue-resident immune cells. In particular, our data first showed a slight decrease in in CD11b⁺ myeloid cell population compared to the young ones. Following the burn injury, CD11b⁺ showed a significant increase both aged and young burn mice (Fig. 5A). On the other hand, we observed significantly fewer F4/80⁺ macrophages, CD 19⁺ B cells, and CD4 + T cells in aged sham mice than their young counterparts (Fig. 6B–D). Furthermore, F4/80⁺ macrophages, CD 19⁺ B, and CD4 + T cells all decreased in young mice following burn injury compared with age-matched sham controls. EV administration only increased the F4/80⁺ macrophage population in aged sham mice but did not induce further significant changes in aged sham or burn mice (Fig. 6B).

This finding suggested that burn trauma affects tissue-resident immune cells in adipose tissue and may influence inflammatory responses which was evident in young mice. In aged mice, burn injury failed to initiate any changes in macrophages or T cells, while B-cell pools were further reduced after burn injury (Fig. 6C,D).

Furthermore, we assumed that aging adipose tissue may exhibit an exhausted T-cell compartment that is further aggravated by burn trauma. Therefore, we next evaluated expression levels of Lag-3 and tim-3. Lag-3 is generally regarded as an exhaustion marker for CD4 + and CD8 + T cells and plays a regulatory role in the immune system, Lag-3 expression is frequently associated with exhausted T cells. Upregulated lag-3 expression leads to the inhibition of cell proliferation, immune function, cytokine secretion, and homeostasis. Interestingly, we observed a significantly upregulated lag-3 expression in aged mice following burn compared to both the aged sham and younger burn mice. Fortunately, lag-3 expression was reduced following EV administration in both the sham and burn mice (Fig. 6E).

Tim-3, another marker of T-cell activation, is produced by inflammatory IFNγ-producing CD4⁺ T cells. Furthermore, Tim-3 is also highly expressed in dysfunctional, exhausted, T cells and leads to progressive loss of effector function. We observed that tim-3 expression is upregulated in both aged sham mice and burn mice and is reduced after EV administration (Fig. 6F). These findings may suggest that aged burn mice exhibit dysregulated inflammatory and dysfunctional T-cell responses, which can be manipulated by EV administration to regulate T-cell fates.

T-cell effector and memory function are restored after EV administration in aged mice

CD44 is a prominent activation marker that distinguishes memory and effector T cells from their naïve counterparts. It also plays a role in early T-cell signaling events, as it is bound to lymphocyte-specific protein kinases and thereby enhances T-cell receptor signaling. There was no change in CD44 expression with age (young vs. aged sham mice) or with burn injury in young mice (Fig. 7A,B). However, CD44 expression is upregulated in aged burn mice. EVs influence distinct responses in aged sham and burn mice; EV administration increased CD44 expression in aged sham mice, while reduced CD44 expression in aged burn mice (Fig. 7A,B). This regulation of CD44 expression is coordinated with CD62L expression; when CD44 is upregulated to mobilize effector T cells at sites of infection and inflammation, L-selectin CD62L expression is downregulated (Fig. 7C,D). Thus, EVs engage the CD44 receptor to regulate effector T cells.

Similarly, CD127 is required for the maintenance, proliferation, and dynamic regulation of T cells. We observed an increase in CD127⁺ cells following the burn injury in both young and aged mice (Fig. 7F), which represents a requirement for maintaining the T-cell pool and function in response to trauma (Fig. 7E,F). CD127 was also increased in aged sham mice after EV administration.

Mitochondrial respiration in the SVF and immune cells

The AT is a highly diverse metabolic organ composed of adipocytes, adipose progenitor cells, and a stromal vascular fraction (SVF) consisting of a heterogeneous cell population, such as mesenchymal progenitor/stem cells, preadipocytes, endothelial cells, pericytes and immune cells. The SVF communicates via an intrinsic signaling cascade that leads to transcriptomic and immunological changes to respond to metabolic demands during stress. In this context, we next analyzed the mitochondrial respiration of the whole SVF and individual immune cells (macrophages, B cells, and T cells). Compared with their aged-matched sham counterparts, SVFs from young burn ATs had increased mitochondrial respiration throughout the assay, including basal respiration (Fig. 8A). Interestingly, respiration in aged sham SVFs in response to the mitochondrial stress assay was greater than that in young sham SVFs. These levels decreased with EV administration and decreased to levels similar to those in young sham SVFs. Importantly, mitochondrial respiration in aged SVFs was blunted after burn injury and was significantly lower than that in young burn SVFs and aged sham SVFs (Fig. 8A,B). These findings indicate that aged mouse SVFs are unable to respond to mitochondrial stress initiated by burn trauma. While basal respiration did not improve in aged burn SVFs, coupling efficiency significantly improved in these mice following EV administration, which was indicative of augmented ATP production (Fig. 8B,C).

We next isolated F4/80⁺ macrophages, CD19⁺ B cells and CD4⁺ T cells from the SVF of ATs and subjected the cells to a mitochondrial stress test. Like those in the SVF, the mitochondrial respiration of macrophages was similar (Fig. 8D). Basal respiration was significantly greater in the young burn MΦ group than in both the young and aged sham MΦ groups (Fig. 8E). Aged burn MΦ had blunted mitochondrial respiration compared to young burn MΦ and aged sham MΦ, further confirming a dysfunctional immunometabolic response. Furthermore, as expected, basal respiration was significantly lower in aged burn MΦ compared to age-matched sham counterparts (Fig. 8E). Interestingly, EV administration significantly improved basal respiration in aged sham MΦ mice however, a similar response was not reproduced in aged burn mice.

Intriguingly, CD19⁺ B cells elicited distinct responses from those of SVF cells and macrophages. No difference was observed in mitochondrial respiration in CD19⁺ B cells between young sham and young burn mice (Fig. 8G). Respiration in response to mitochondrial stress was greater in aged sham CD19⁺ B cells than in control CD19 + B cells and further increased in response to burn injury in aged burn CD19⁺ B cells. Intriguingly, mitochondrial respiration significantly decreased after EV administration to aged burn-treated CD19⁺ B cells. Additionally, the coupling efficiency was significantly lower for aged burn CD19⁺ B cells than for young burn CD19⁺ B cells, and this effect was modestly improved by EV administration (Fig. 8H,I).

Finally, similar to the whole SVF and macrophages, CD4⁺ T cells are more hyperrespiratory following burn trauma in young AT compared to shams. Counterintuitively, the CD4⁺ T cells in the aged burn group were also hyperrespiratory and exhibited responses very similar to those of young burn CD4⁺ T cells (Fig. 8J). This finding suggested that T cells do not become metabolically dysfunctional with age and respond to burn trauma by becoming hyperrespiratory. However, whether this increase in mitochondrial respiration in response to burn injury is beneficial or detrimental has yet to be determined. EV administration reduced total mitochondrial respiration throughout the assay, decreased basal respiration and improved coupling efficiency in CD4⁺ T cells in the aged burn group, indicating a possible therapeutic advantage (Fig. 8K,L).

These results suggest that immunometabolic responses of the SVF, while performing as a distinctive compartment of the AT, are rather driven by individual immune cells with each immunophenotype serving a distinct function in response to burn trauma and therefore warrants a further in depth investigation.

Lipolysis is mitigated following EV administration in aged mice

We next examined the impact of EV administration on lipolysis in aged mice. For this purpose, we assessed adipose triglyceride lipase (ATGL), a triacylglycerol lipase responsible for the breakdown of fat stores into free fatty acids (FFAs) and glycerol. Chronological aging is associated with increased adipose lipolysis and lipid metabolism. Intuitively, aged sham mice exhibited the highest levels of ATGL, which were significantly reduced after EV administration. Interestingly, ATGL levels were significantly lower in aged burn ATs than in control ATs and further reduced after EV administration (Fig. 9A,B). Similarly, we assessed the expression of hormone sensitive lipase (HSL), another key enzyme involved in lipid metabolism. As expected, EV administration reduced the p-HSL/total HSL levels in the aged sham and burn mice (Fig. 9A,C). This finding is promising since lipid metabolism plays a central role in burn-induced AT dysfunction, and a decrease in circulating fatty acids following EV administration can mitigate burn‐induced hepatomegaly.

Aging and burn injury induces fatty infiltration in the liver, which is rescued by EVs

Adipose tissue lipolysis leads to increased systemic free fatty circulation in other organs, such as the liver. We observed an increase in the deposition of fat droplets in the livers of aged sham and aged burn mice (Fig. 9D). Fortunately, free fatty acid infiltration in the liver was reduced with EV administration in both sham-treated and burn-aged mice. Furthermore, while chronological aging itself did not increase liver mass (young sham vs. aged sham), burn injury resulted in a more pronounced and significant increase in liver mass in young mice than in their aged counterparts (Fig. 9E). Interestingly, EV administration resulted in increased liver mass in aged sham and burn mice. We next measured the levels of serum FFA and observed an increase in the levels of these fatty acids in young mice postburn compared to those in young sham mice (Fig. 9F). Intuitively, EV administration reduced FFA levels in both aged sham and burn mice. Similarly, serum triglyceride levels were significantly greater following burn injury in both young and aged mice than in the respective age-matched sham controls. Furthermore, triglyceride (TG) levels were significantly greater in aged burn mice than in young burn mice (Fig. 9G). Consequently, EV administration produced a modest decrease in TG levels in aged sham and aged burn mice.

The effect of EV administration on hepatocellular signaling

Next, we further investigated the effect of EV administration on hepatocellular signaling pathways, which are crucial for the regulation of hepatic inflammation, fat accumulation, and dysfunction. We measured the protein expression levels of total and phosphorylated Akt and observed significant increases in the p-Akt/total Akt levels in the livers of both aged sham and burn mice following EV administration (Fig. 10A,B). Other reports have shown reduced p-Akt and increased TG levels in high-fat diet groups³⁴. Our results corroborate the decreased TG levels and indicate reduced fat mass accumulation in the liver after EV administration. It is well established that hepatic Akt is essential for maintaining whole-body glucose homeostasis and insulin sensitivity and regulates diverse cellular events, including hepatocyte survival and apoptosis, while inhibition of Akt signaling may lead to hepatocellular injury through activation of the mitochondrial membrane pathway of apoptosis. Similarly, the MAPK ERK signaling pathway is activated mainly by mitogens and growth factor signals and plays an important role in mediating hepatic inflammation. We observed an increase in p-ERK/total ERK protein levels in the livers of aged sham and burn mice following EV administration (Fig. 10A,C).

Finally, we investigated whether EV administration could rescue the endoplasmic reticulum (ER) and ameliorate oxidative stress. Notably, phosphorylation of the eukaryotic translation initiation factor 2 alpha subunit (eIF2α) initiates adaptive gene expression to restore homeostasis following trauma. Specifically, eIF2α phosphorylation is required to prevent hepatic fat infiltration, hepatocyte death, and liver fibrosis. We observed an increase in p-EIF2a/total EIF2a protein levels in the livers of aged sham and burn mice following EV administration (Fig. 10A,D).

Discussion

The EV cargo is highly characteristic of its parent cell source and contains bioactive proteins, nucleic acids, lipids and cellular metabolites. EVs are implicated in cell-to-cell communication and mediate cellular signaling by functionally impacting recipient cells through the transport of diverse cargo. In addition to changes in EV cargo with chronological aging, several recent studies have highlighted the role of EVs in metabolism. Thus, EVs can act as cellular messengers to reprogram the metabolic machinery in recipient cells and thus influence systemic pathophysiological responses.

In this study, we compared the response of young and aged mice to burn injury and subsequent alterations in adipose tissue function and metabolism. We further investigated whether intravenous EV administration could ameliorate systemic dysfunction to near physiological levels. Cellular senescence is an established hallmark of chronological aging; consistent with this, we observed increased senescence in the adipose tissue of aged mice. Surprisingly, burn trauma further significantly increased senescence in aged adipose tissue, which was attenuated by the administration of EVs from young mouse serum. This finding corroborates the significantly increased expression of miR-146a, miR‐223, and miR‐145 in EVs from aged sera compared to young sera and may have driven the reduction in the senescent phenotype in adipose tissue following the administration of EVs from young sera. EVs have been shown to induce antisenescence by activating the IGF1/PI3K/AKT pathway³⁵. Similarly, we observed an increase in p-AKT levels in the liver following EV administration. This finding suggested that the cargo of young EVs can rejuvenate adipose tissue and reduce the senescence burden by clearing senescent cells within adipose tissue⁹ and influencing their function³⁶.

We have shown that adipose tissue is a master regulator of systemic metabolism and heavily influences postburn outcomes. Studies have also shown that adipose tissue regulates systemic aging through EV-mediated mechanisms^37,38. Interestingly, EVs also mediate interorgan signals, and in particular, EVs constitute an essential part of the human adipose secretome^39,40. A recent study showed that plasma extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is carried by EVs and that its levels are decreased in older mice and elderly individuals. EVs containing eNAMPT induce NAD⁺ biosynthesis and significantly extend the lifespan of aged mice⁴¹. To this end, adipose tissue browning is impaired during aging, while normalizing adipose tissue [NAD⁺/ NADH] recovers beige adipocytes. This finding is consistent with our finding that we observed increased expression of the master browning marker UCP-1 after EV administration, representing a phenotypic switch from white to beige adipose tissue. NAD⁺ is required for a robust metabolic response; however, while it will be interesting to measure intracellular [NAD⁺/NADH] levels, we did observe an enhanced metabolic response following EV administration.

To this end it may be presumed that exogenous systemic EV administration to aged subjects may initiate a positive feedback loop to improve systemic metabolism, and each organ influences the metabolic status of the other organ, as we observed in the case of adipose tissue, liver, and the immune cell compartment. Therefore, it may be hypothesized that exogenous EV administration may influence the cargo of tissue-specific EVs, such as adipose-derived EVs. Adipocyte-derived EVs have been shown to be mediators of insulin resistance in the liver^42,43, regulate adipose tissue inflammation by recruiting monocytes and can also be transferred from adipose tissue endothelial cells to adipocytes and vice versa in vivo⁴⁴. In this regard, EVs derived from beige adipocytes have been shown to ameliorate hepatic steatosis and improve glucose tolerance⁴⁵. Intriguingly, adipocyte-derived EVs constitute the majority of systemic circulating EVs⁴², and white and beige adipocytes are distinct.

Chronological aging has several implications for burn trauma; not only is the cargo of EVs distinct between younger and aged counterparts, but aged adipose tissue also fails to morphologically and phenotypically convert to beige/brown adipocytes due to reduced intracellular cAMP levels and thus fails to initiate systemic responses to burn injury, which are required for organ function and survival. In addition, we observed a 2-fold decrease in the miR-126 concentration in aged vs. young EVs. A reduction in miR-126 leads to the inhibition of the Erk1/2 mitogen‐activated protein kinase (MAPK) pathway, which impairs the angiogenic ability of the organ⁴⁶. Like in obesity, we observed overexpression of miRNA-155 in EVs from aged sera. miRNA-155 heavily influences the inflammatory phenotype of macrophages and regulates adipose tissue homeostasis through direct suppression of its targets, the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein β (CEBPβ)⁴⁷. Finally, since myeloid cells are major targets of circulating EVs⁴⁸, we assessed the effect of EV administration on the immune cell compartment of adipose tissue. Aged EVs had greater expression of miR‐146a, miR‐145, and miR‐223. Mechanistically, key signal transduction factors in the MAPK⁴⁹ and AKT⁵⁰ pathways are targeted by these miRNAs to regulate cellular responses to stimuli. This finding suggested that the cellular expression of these microRNAs in aged subjects may be responsible for the blunted response of immune cells to burn trauma, as shown by mitochondrial respiration. As observed in NAFLD, hepatocyte apoptosis is correlated with progressive inflammation and fibrosis⁵¹ and is linked to mitochondrial dysfunction⁵². Mitochondria regulate both cell proliferation and death, including apoptosis⁵³, and mediate the PI3K/Akt signaling pathway. Consistent with these findings, we observed increased expression of p-AKT and p-ERK in the liver after EV administration, highlighting the role of EVs in rescuing the liver from postburn hepatocyte apoptosis and hepatic steatosis.

The effect of exogenous EVs on tissue energy consumption and the utilization machinery following burn injury has yet to be determined and is an interesting avenue to explore. Exosomal metabolites act as lipid facilitators in intercellular transport⁵⁴. Furthermore, glutamate contained within EVs might drive carbon and nitrogen trafficking⁵⁵, while lactate might help in low-oxygen environments⁵⁶. EVs have also been shown to restore glycolysis in injured organs since they contain fully active glycolytic enzymes⁵⁷. Intriguingly, we observed that treatment with mitochondrial complex inhibitors did not significantly reduce oxygen consumption in the immune cell populations under investigation. This suggests that a substantial portion of oxygen consumption in these immune cells may not be driven by classical mitochondrial respiration. Immune cells particularly when activated by injury or inflammatory cues undergo metabolic reprogramming that shifts their reliance away from mitochondrial oxidative phosphorylation and toward glycolysis and alternative oxygen-consuming pathways^58,59,60. In this activated state, immune cells upregulate enzymes such as NADPH oxidases (NOX), cyclooxygenases (COX), and lipoxygenases (LOX), which consume oxygen independently of the electron transport chain and are thus unaffected by rotenone or antimycin inhibition^61,62. This shift supports immune functions such as reactive oxygen species (ROS) production and inflammatory cytokine release. To this end, future experiments should aim to dissect the sources of non-mitochondrial oxygen consumption in immune cells under tissue injury or disease states by including additional inhibitors such as diphenyleneiodonium (DPI) to block NADPH oxidases, or COX/LOX inhibitors, and by measuring ROS production, glycolytic flux, and metabolic gene expression profiles. Pairing Seahorse analysis with immunometabolic assays and transcriptional profiling will help clarify the relative contributions of mitochondrial versus non-mitochondrial pathways to total OCR in activated immune cells.

Finally, EVs are also produced by the adipose tissue itself which play crucial roles in metabolic homeostasis and in disease states. Adipose-derived EVs function as endocrine messengers, enabling long-distance communication between adipose tissue and other organs such as the liver, muscle, pancreas, and brain by transporting their bioactive cargos. The cargo composition reflects the physiological condition of the adipose tissue, changing in response to factors like nutritional state and adipocyte health. How this cargo is alters in burn-induced hypermetabolism needs to be further explored which will open new avenues for therapeutic interventions. For instance, these EVs carry pro-inflammatory cytokines and dysregulated miRNAs that contribute to insulin resistance and metabolic dysfunction across multiple organs. They promote hepatic insulin resistance and lipid accumulation, impair muscle insulin sensitivity and mitochondrial activity, influence pancreatic β-cell survival and insulin secretion, and modulate immune responses through effects on macrophage polarization. Given this broad functional impact, adipose-derived EVs show potential as non-invasive biomarkers and as vehicles for therapeutic intervention. Furthermore, their effects are depot- and cell-type specific, with distinct signaling profiles observed between subcutaneous vs. visceral fat and white vs. brown adipocytes, emphasizing their nuanced roles in inter-tissue communication.

Conclusion

We have shown that AT is a master regulator of systemic metabolism that strongly influences postburn outcomes. AT regulates systemic aging through EV-mediated mechanisms^37,38. We demonstrated that treatment with EVs from young serum samples has the potential to ameliorate AT dysfunction postburn, restore mitochondrial bioenergetics and improve immune cell infiltration and immuno-metabolic function in older burn patients, thus improving postburn outcomes and survival in this vulnerable population.

To this end, in our study we explored the potential of blood serum-derived EVs. It is worthy to note that the clotting process in the EV isolation approach may alter the EV levels by inducing their release from platelets or other cells in blood or by degradation of EVs. Thus, whether the observed effects are from platelet-derived EVs will be elucidated in subsequent studies. Further, for future studies, it is important to investigate plasma EVs in which plasma is obtained by adding anticoagulants to blood samples, preventing clotting and thus preserving the native state of sampled EVs.

Materials and methods

EV isolation

Whole blood was collected from 8- to 12-week-old mice via cardiac puncture following euthanasia. Blood was allowed to clot at room temperature, and the serum was separated via centrifugation. One milliliter of serum was pooled to constitute EVs per intravenous injection. EVs were collected using Cell Guidance Systems Exo-spin™ mini columns following the manufacturer’s instructions. EV fractions were concentrated using 100 kDa filters. The final serum EVs were used in all experiments.

TEM

The EVs were diluted to a concentration of 1 mg/ml, and 4 µl was deposited onto a glow-discharged copper grid positioned on filter paper. Afterward, the grid was dried for 20 min using an infrared lamp. The grid was subjected to a triple wash procedure to eliminate any residual buffer salts that could manifest as opaque crystals via transmission electron microscopy. Distilled water was pipetted onto the grid, followed by removal of the water using filter paper. Following the washing steps, the grid was stained with a 2% solution of uranyl acetate at a pH of 7.0 for 40 s. Subsequently, the grid was left to air-dry at room temperature. Exosomes were then examined using a high-resolution transmission electron microscope (H-650 Hitachi microscope, Japan) operating at 80 kV.

MicroRNA analysis

EVs were isolated as described above. miRNAs were isolated and profiled using a Systems Bio Mouse Complete SeraMir Exosome RNA Amplification & Profiling Kit following the manufacturer’s instructions. miRNA expression in aged serum samples was compared against that in young serum samples. miEAA-miRNA enrichment and annotation analysis were conducted using the miRNA Enrichment Analysis and Annotation Tool (miEAA).

Animals

All vertebrate animal experiments were conducted according to ethics and ARRIVE guidelines and were approved by the Sunnybrook Research Institute Animal Care Committee (protocol number 21467). All experiments were performed in accordance with relevant guidelines and regulations. All procedures involving animals were performed in compliance with the Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care (CACC). C57BL/J6 mice aged 78–80 weeks were purchased from The Jackson Laboratory (JAX^®). Mice were kept in temperature-controlled cages according to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and fed ad libitum. Mice were acclimated for 1–2 weeks prior to the experiments.

Mouse burn injury model

We used a well-established 20% TBSA full-thickness burn model (47). Mice were anesthetized using 2.5% isoflurane, after which the ventral surface and dorsal spine region were shaved. To protect the spine, 2 mL of Ringer’s lactate was injected subcutaneously prior to the burn wound. For pain control, 0.05–0.1 mg/kg body weight buprenorphine was injected before and after burn injury. A full-thickness third-degree scald burn was induced by placing the mouse in a mold that was exposed to 20% TBSA and immersing the mold in a 98 °C water bath for 10 s. Mice were then housed individually for health score assessment until sacrifice. Tissues, organs, and blood samples were collected upon sacrifice and processed immediately or stored at -80 °C until analysis.

Euthanasia

Euthanasia was performed according to Canadian Council on Animal Care (CCAC) guidelines. Briefly, animals were anesthetized, and cervical dislocation was performed. Cervical dislocation was confirmed by palpation of the vertebrae and adequate separation was confirmed.

SVF isolation

Adipose tissue was collected from burn and sham mice. First, the tissue was cut into small pieces (~ 0.5 cm) and digested in DMEM + 1 mg/mL collagenase type II (Sigma) for 40 min with shaking at 37 °C. The cell suspensions were subsequently filtered through a 100 μm cell strainer and centrifuged. The floating adipocyte layer and SVF pellet were collected separately in complete DMEM (+ 10% fetal bovine serum - FBS) (48). The SVF was either stained for flow cytometry or subjected to magnetic activated cell sorting (MACS).

Magnetic activated cell sorting (MACS)

SVF was used to sort macrophages, B cells and T cells using Miltenyi Biotec macrophage (130-110-434), Pan B-cell (130-104-443), and Pan T-cell (130-095-130) isolation kits.

Flow cytometry

SVFs were resuspended in PBS + 2% FBS. Surface markers were stained with monoclonal antibodies for multicolor flow cytometry (Table 1) diluted in PBS + 2% FBS (1:200) for 20 min. Cells were fixed with 4% paraformaldehyde (PFA) solution for 20 min. Cells were washed in PBS + 2% FBS, acquired on a BD LSRII using BD FACSDiva software (BD Biosciences) and analyzed with FlowJo Software (BD Biosciences). Gating strategy is reported in Supplementary Figs. 2–4.

Table 1 List of antibodies used for flow cytometry.

Full size table

Quantitative PCR (qPCR)

Total RNA was isolated from whole adipose tissue using TRI Reagent (Sigma) according to the manufacturer’s instructions. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time quantitative PCR was performed using RT-Real-Time iTaq™ Universal SYBR^® Green Supermix (Bio-Rad) with specific primers. Gene expression was calculated through the formula ΔΔCt = ΔCt of test sample - ΔCt of control sample (young shams), where ΔCt = Ct gene studied – Ct 18 s (housekeeping gene, used as an internal control for the reaction).

Western blot

Tissues were lysed for immunoblotting in 1% DDM lysis buffer [1% DDM in 10 µM ML211, Dulbecco’s phosphate-buffered saline (DPBS), protease inhibitor cocktail (1x), phosphatase inhibitor cocktail 2 (1:100) and PMSF (10 mM)]. Afterwards, a Bio-Rad Bradford protein assay was used to measure the protein concentration, where equal volumes of protein were then loaded for immunoblot analysis. Afterwards, 30 µg of protein lysates were separated via SDS‒PAGE and transferred to nitrocellulose membranes (Bio-Rad). Nitrocellulose membranes were then blocked in 5% bovine serum albumin (BSA) in PBS-T (0.1% Tween-20 in PBS). Blocking was performed for 1 h at room temperature, after which the sections were incubated with primary antibodies at a 1:1000 dilution overnight in a 4 °C refrigerator. After primary incubation, the membranes were incubated with anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase. The secondary incubation took 1 h at room temperature and was followed by three washes in PBS-T. The nitrocellulose membranes were then imaged with the use of an enhanced chemiluminescence (ECL) substrate (Bio-Rad) and the ChemiDoc™ MP System (Bio-Rad). Finally, the proteins were analyzed using ImageJ software to quantify the band intensity and calculate the absorbance ratio of β-actin.

Seahorse assay

For real-time analysis of the oxygen consumption rate (OCR), mitochondria were subjected to a mitochondrial stress test on an XFe96 Extracellular Flux Analyzer (Agilent) according to the manufacturer’s instructions. Three or more consecutive measurements were obtained under baseline conditions and after injection of specific metabolic modulators: 1 µmol/L oligomycin, 1.5 µmol/L fluoro-carbonyl cyanide phenylhydrazone (FCCP), and 100 nmol/L rotenone + 1 µmol/L antimycin A (all Sigma reagents). The data were analyzed using Seahorse XF Wave software. For isolated SVFs or MAC-sorted macrophages, B cells or T cells, 100,000-200,000 cells were plated in each well and subjected to a mitochondrial stress test as above. The OCRs were normalized to the cell number × 10⁶.

Data analysis

Data were analyzed with Student’s t test or one-way ANOVA or two-way ANOVA followed by Tukey’s test or the Fisher’s LSD test to determine the significance of the differences. Values were considered significant at p < 0.05. All the data were analyzed with Prism 9 (GraphPad) and are shown as the mean ± standard error of the mean (SEM).

Data availability

Data can be made available from the corresponding author on reasonable request.

References

Llompart-Pou, J. A., Perez-Barcena, J., Chico-Fernandez, M., Sanchez-Casado, M. & Raurich, J. M. Severe trauma in the geriatric population. World J. Crit. Care Med. 6, 99–106. https://doi.org/10.5492/wjccm.v6.i2.99 (2017).
Article PubMed PubMed Central Google Scholar
Idrovo, J. P. et al. Advanced age heightens hepatic damage in a murine model of scald burn injury. J. Trauma. Acute Care Surg. 90, 731–737. https://doi.org/10.1097/TA.0000000000003048 (2021).
Article CAS PubMed PubMed Central Google Scholar
Jeschke, M. G. et al. Pathophysiologic response to burns in the elderly. EBioMedicine 2, 1536–1548 https://doi.org/10.1016/j.ebiom.2015.07.040 (2015).
Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217. https://doi.org/10.1016/j.cell.2013.05.039 (2013).
Article CAS PubMed PubMed Central Google Scholar
Schaum, N. et al. Ageing hallmarks exhibit organ-specific Temporal signatures. Nature 583, 596–602. https://doi.org/10.1038/s41586-020-2499-y (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Schosserer, M., Grillari, J., Wolfrum, C. & Scheideler, M. Age-Induced changes in white, brite, and brown adipose depots: A mini-review. Gerontology 64, 229–236. https://doi.org/10.1159/000485183 (2018).
Article CAS PubMed Google Scholar
Sidossis, L. S. et al. Browning of subcutaneous white adipose tissue in humans after severe adrenergic stress. Cell. Metab. 22, 219–227. https://doi.org/10.1016/j.cmet.2015.06.022 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wang, W. et al. Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance. FASEB J 32, fj201800059R https://doi.org/10.1096/fj.201800059R (2018).
Fafian-Labora, J. A., Rodriguez-Navarro, J. A. & O’Loghlen, A. Small extracellular vesicles have GST activity and ameliorate senescence-related tissue damage. Cell Metab 32, 71–86 e75 https://doi.org/10.1016/j.cmet.2020.06.004 (2020).
Mehta, A. & Baltimore, D. MicroRNAs as regulatory elements in immune system logic. Nat. Rev. Immunol. 16, 279–294. https://doi.org/10.1038/nri.2016.40 (2016).
Article CAS PubMed Google Scholar
Paladini, L. et al. Targeting MicroRNAs as key modulators of tumor immune response. J. Exp. Clin. Cancer Res. 35, 103. https://doi.org/10.1186/s13046-016-0375-2 (2016).
Article CAS PubMed PubMed Central Google Scholar
O’Connell, R. M., Rao, D. S., Chaudhuri, A. A. & Baltimore, D. Physiological and pathological roles for MicroRNAs in the immune system. Nat. Rev. Immunol. 10, 111–122. https://doi.org/10.1038/nri2708 (2010).
Article CAS PubMed Google Scholar
Datto, M. B., Yu, Y. & Wang, X. F. Functional analysis of the transforming growth factor beta responsive elements in the WAF1/Cip1/p21 promoter. J. Biol. Chem. 270, 28623–28628. https://doi.org/10.1074/jbc.270.48.28623 (1995).
Article CAS PubMed Google Scholar
Dews, M. et al. The myc-miR-17 ~ 92 axis blunts TGFbeta signaling and production of multiple TGFbeta-dependent antiangiogenic factors. Cancer Res. 70, 8233–8246. https://doi.org/10.1158/0008-5472.CAN-10-2412 (2010).
Article CAS PubMed PubMed Central Google Scholar
Ohgushi, M. et al. Transforming growth factor beta-dependent sequential activation of smad, bim, and caspase-9 mediates physiological apoptosis in gastric epithelial cells. Mol. Cell. Biol. 25, 10017–10028. https://doi.org/10.1128/MCB.25.22.10017-10028.2005 (2005).
Article CAS PubMed PubMed Central Google Scholar
Zaidi, S. K. et al. Altered Runx1 subnuclear targeting enhances myeloid cell proliferation and blocks differentiation by activating a miR-24/MKP-7/MAPK network. Cancer Res. 69, 8249–8255. https://doi.org/10.1158/0008-5472.CAN-09-1567 (2009).
Article CAS PubMed PubMed Central Google Scholar
Kong, K. Y. et al. MIR-23A microRNA cluster inhibits B-cell development. Exp Hematol 38, 629–640 e621 https://doi.org/10.1016/j.exphem.2010.04.004 (2010).
Kurkewich, J. L. et al. The miR-23a ~ 27a ~ 24 – 2 MicroRNA cluster buffers transcription and signaling pathways during hematopoiesis. PLoS Genet. 13, e1006887. https://doi.org/10.1371/journal.pgen.1006887 (2017).
Article CAS PubMed PubMed Central Google Scholar
Ben-Ami, O., Pencovich, N., Lotem, J., Levanon, D. & Groner, Y. A regulatory interplay between miR-27a and Runx1 during megakaryopoiesis. Proc. Natl. Acad. Sci. U S A. 106, 238–243. https://doi.org/10.1073/pnas.0811466106 (2009).
Article ADS PubMed Google Scholar
Mavrakis, K. J. et al. A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat. Genet. 43, 673–678. https://doi.org/10.1038/ng.858 (2011).
Article CAS PubMed PubMed Central Google Scholar
Wang, G. et al. miR-23a suppresses proliferation of osteosarcoma cells by targeting SATB1. Tumour Biol. 36, 4715–4721. https://doi.org/10.1007/s13277-015-3120-0 (2015).
Article CAS PubMed Google Scholar
Taganov, K. D., Boldin, M. P., Chang, K. J. & Baltimore, D. NF-kappaB-dependent induction of MicroRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. U S A. 103, 12481–12486. https://doi.org/10.1073/pnas.0605298103 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
He, Y. et al. MiR-146a regulates IL-6 production in lipopolysaccharide-induced RAW264.7 macrophage cells by inhibiting Notch1. Inflammation 37, 71–82. https://doi.org/10.1007/s10753-013-9713-0 (2014).
Article CAS PubMed Google Scholar
Self-Fordham, J. B., Naqvi, A. R., Uttamani, J. R., Kulkarni, V. & Nares, S. MicroRNA: dynamic regulators of macrophage polarization and plasticity. Front. Immunol. 8, 1062. https://doi.org/10.3389/fimmu.2017.01062 (2017).
Article CAS PubMed PubMed Central Google Scholar
Hou, J. et al. MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J. Immunol. 183, 2150–2158. https://doi.org/10.4049/jimmunol.0900707 (2009).
Article CAS PubMed Google Scholar
Mohnle, P. et al. MicroRNA-146a controls Th1-cell differentiation of human CD4 + T lymphocytes by targeting PRKCepsilon. Eur. J. Immunol. 45, 260–272. https://doi.org/10.1002/eji.201444667 (2015).
Article CAS PubMed Google Scholar
Huffaker, T. B. et al. Epistasis between MicroRNAs 155 and 146a during T cell-mediated antitumor immunity. Cell. Rep. 2, 1697–1709. https://doi.org/10.1016/j.celrep.2012.10.025 (2012).
Article CAS PubMed PubMed Central Google Scholar
Dudda, J. C. et al. MicroRNA-155 is required for effector CD8 + T cell responses to virus infection and cancer. Immunity 38, 742–753. https://doi.org/10.1016/j.immuni.2012.12.006 (2013).
Article CAS PubMed PubMed Central Google Scholar
Mann, M. et al. An NF-kappaB-microRNA regulatory network tunes macrophage inflammatory responses. Nat. Commun. 8, 851. https://doi.org/10.1038/s41467-017-00972-z (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Abdullahi, A., Knuth, C.M., Auger, C., Sivayoganathan, T., Parousis, A. & Jeschke, M.G. Adipose browning response to burn trauma is impaired with aging. p.e143451. JCI insight 6 (16), e143451 (2021).
Auger, C., Samadi, O. & Jeschke, M. G. The biochemical alterations underlying post-burn hypermetabolism. Biochim. Biophys. Acta Mol. Basis Dis. 1863, 2633–2644. https://doi.org/10.1016/j.bbadis.2017.02.019 (2017).
Article CAS PubMed Google Scholar
Auger, C. et al. Metformin prevents the pathological Browning of subcutaneous white adipose tissue. Mol. Metab. 29, 12–23. https://doi.org/10.1016/j.molmet.2019.08.011 (2019).
Article CAS PubMed PubMed Central Google Scholar
Abdullahi, A. & Jeschke, M. G. Taming the flames: targeting white adipose tissue Browning in hypermetabolic conditions. Endocr. Rev. 38, 538–549. https://doi.org/10.1210/er.2017-00163 (2017).
Article PubMed PubMed Central Google Scholar
Han, J. W. et al. Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats. World J. Gastroenterol. 16, 6111–6118. https://doi.org/10.3748/wjg.v16.i48.6111 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, Y. et al. Embryonic stem cell-derived extracellular vesicles enhance the therapeutic effect of mesenchymal stem cells. Theranostics 9, 6976–6990. https://doi.org/10.7150/thno.35305 (2019).
Article CAS PubMed PubMed Central Google Scholar
Alibhai, F. J. et al. Cellular senescence contributes to age-dependent changes in Circulating extracellular vesicle cargo and function. Aging Cell. 19, e13103. https://doi.org/10.1111/acel.13103 (2020).
Article CAS PubMed PubMed Central Google Scholar
Yoon, M. J. et al. SIRT1-Mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD + and function in mice. Cell. Metab. 21, 706–717. https://doi.org/10.1016/j.cmet.2015.04.002 (2015).
Article CAS PubMed PubMed Central Google Scholar
Imai, S. Nicotinamide phosphoribosyltransferase (Nampt): a link between NAD biology, metabolism, and diseases. Curr. Pharm. Des. 15, 20–28. https://doi.org/10.2174/138161209787185814 (2009).
Article CAS PubMed PubMed Central Google Scholar
Hartwig, S. et al. Exosomal proteins constitute an essential part of the human adipose tissue secretome. Biochim. Biophys. Acta Proteins Proteom. 1867, 140172. https://doi.org/10.1016/j.bbapap.2018.11.009 (2019).
Article CAS PubMed Google Scholar
Gesmundo, I. et al. Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic beta cells. JCI Insight. 6 https://doi.org/10.1172/jci.insight.141962 (2021).
Yoshida, M. et al. Extracellular vesicle-contained eNAMPT delays aging and extends lifespan in mice. Cell Metab 30, 329–342 e325 https://doi.org/10.1016/j.cmet.2019.05.015 (2019).
Thomou, T. et al. Adipose-derived Circulating MiRNAs regulate gene expression in other tissues. Nature 542, 450–455. https://doi.org/10.1038/nature21365 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Koeck, E. S. et al. Adipocyte exosomes induce transforming growth factor beta pathway dysregulation in hepatocytes: a novel paradigm for obesity-related liver disease. J. Surg. Res. 192, 268–275. https://doi.org/10.1016/j.jss.2014.06.050 (2014).
Article CAS PubMed Google Scholar
Crewe, C. et al. An Endothelial-to-adipocyte extracellular vesicle axis governed by metabolic state. Cell 175, 695–708 e613 https://doi.org/10.1016/j.cell.2018.09.005 (2018).
Jung, Y. J. et al. Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. Sci. Adv. 6, eaay6721. https://doi.org/10.1126/sciadv.aay6721 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Togliatto, G. et al. Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications. Int. J. Obes. (Lond). 40, 102–111. https://doi.org/10.1038/ijo.2015.123 (2016).
Article CAS PubMed Google Scholar
Chen, Y. et al. miR-155 regulates differentiation of brown and beige adipocytes via a bistable circuit. Nat. Commun. 4, 1769. https://doi.org/10.1038/ncomms2742 (2013).
Article ADS CAS PubMed Google Scholar
Akbar, N. et al. Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction. JCI Insight 2 https://doi.org/10.1172/jci.insight.93344 (2017).
Cheng, H. S. et al. MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways. EMBO Mol. Med. 5, 1017–1034. https://doi.org/10.1002/emmm.201202318 (2013).
Article CAS PubMed Google Scholar
Meng, F. et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133, 647–658. https://doi.org/10.1053/j.gastro.2007.05.022 (2007).
Article CAS PubMed Google Scholar
Malhi, H. & Gores, G. J. Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease. Semin Liver Dis. 28, 360–369. https://doi.org/10.1055/s-0028-1091980 (2008).
Article CAS PubMed PubMed Central Google Scholar
Caldwell, S. H. et al. Intramitochondrial crystalline inclusions in nonalcoholic steatohepatitis. Hepatology 49, 1888–1895. https://doi.org/10.1002/hep.22851 (2009).
Article PubMed Google Scholar
Lee, D. H., Szczepanski, M. J. & Lee, Y. J. Magnolol induces apoptosis via inhibiting the EGFR/PI3K/Akt signaling pathway in human prostate cancer cells. J. Cell. Biochem. 106, 1113–1122. https://doi.org/10.1002/jcb.22098 (2009).
Article CAS PubMed Google Scholar
Zakharova, L., Svetlova, M. & Fomina, A. F. T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor. J. Cell. Physiol. 212, 174–181. https://doi.org/10.1002/jcp.21013 (2007).
Article CAS PubMed Google Scholar
Hannafon, B. N. & Ding, W. Q. Intercellular communication by exosome-derived MicroRNAs in cancer. Int. J. Mol. Sci. 14, 14240–14269. https://doi.org/10.3390/ijms140714240 (2013).
Article CAS PubMed PubMed Central Google Scholar
Vallabhaneni, K. C. et al. Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microrna, proteins, and metabolites. Oncotarget 6, 4953–4967. https://doi.org/10.18632/oncotarget.3211 (2015).
Article PubMed Google Scholar
Zhang, Y., Liang, F., Zhang, D., Qi, S. & Liu, Y. Metabolites as extracellular vesicle cargo in health, cancer, pleural effusion, and cardiovascular diseases: an emerging field of study to diagnostic and therapeutic purposes. Biomed. Pharmacother. 157, 114046. https://doi.org/10.1016/j.biopha.2022.114046 (2023).
Article CAS PubMed Google Scholar
Mills, E. L. et al. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell 167 (2), 457–470e13 (2016).
Article CAS PubMed PubMed Central Google Scholar
Cheng, S. C. et al. mTOR- and HIF-1α–mediated aerobic Glycolysis as metabolic basis for trained immunity. Science 345 (6204), 1250684 (2014).
Article PubMed PubMed Central Google Scholar
Tannahill, G. M. et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature 496 (7444), 238–242 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N. & Jastroch, M. Analysis and interpretation of microplate-based oxygen consumption and pH data. Nat. Protoc. 9 (2), 182–211 (2014).
Google Scholar
Van der Windt, G. J., Chang, C. H. & Pearce, E. L. Measuring bioenergetics in T cells using the seahorse extracellular flux analyzer. Nat. Protoc. 8 (7), 1472–1485 (2013).
Google Scholar

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) Grant Number R01GM133961. We also thank the Juravinski Research Foundation for their generous support.

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Authors and Affiliations

Department of Surgery, McMaster University, Hamilton, ON, Canada
Ayesha Aijaz, Shayahati Bieerkehazhi, Lauar de Brito Monteiro & Marc G. Jeschke
Center for Burn Research, Hamilton Health Sciences, Hamilton, ON, Canada
Ayesha Aijaz, Shayahati Bieerkehazhi, Lauar de Brito Monteiro, Sean Saladhna, Bhavishya Challagundla, Graham Rix & Marc G. Jeschke
Institute of Medical Science, University of Toronto, Toronto, ON, Canada
Sara Faour, Faraz Chogan & Marc G. Jeschke
Sunnybrook Research Institute, Toronto, ON, Canada
Navneet Kang

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Ayesha Aijaz
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AA designed and performed the experiments, analyzed the data, and wrote the manuscript. LBM conducted flow cytometry and analyzed data. SB conducted western blot. SB, NK, LBM, SS, SF, BC, GR and FC performed the experiments and edited the manuscript. MGJ designed the experimental outlines and edited the manuscript.

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Correspondence to Marc G. Jeschke.

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Aijaz, A., Bieerkehazhi, S., Kang, N. et al. Young extracellular vesicles restore burn-induced adipose tissue immunometabolic and mitochondrial function in older mice. Sci Rep 15, 35328 (2025). https://doi.org/10.1038/s41598-025-19239-5

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Received: 20 January 2025
Accepted: 08 September 2025
Published: 09 October 2025
DOI: https://doi.org/10.1038/s41598-025-19239-5