Distinct immunomodulation elicited by young versus aged extracellular vesicles in bone marrow-derived macrophages

Abstract

Background: Previous research has indicated that extracellular vesicles (EVs) potentially play significant roles in multiple ageing phenotypes. This study uses a factorial experimental design to explore the interactions between circulating EVs and bone marrow-derived macrophages (BMDMs) isolated from young (7-12 weeks) and aged (70-90 weeks) mice.

Results: In this study, plasma EVs from young (Y_EV) and aged (O_EV) mice were isolated and compared based on abundance, size, and miRNA cargo. Compared to some previous studies, we found relatively few differences in EV miRNA cargo between Y_EVs and O_EVs. Young and old EVs were then used to stimulate naïve BMDMs isolated from young (Y_BMDM) and aged (O_BMDM) mice. A panel of five "M1" and six "M2" macrophage markers were used to assess the degree of polarisation. Our results revealed differences in the immunomodulatory effects of Y_EVs and O_EVs in Y_BMDMs and O_BMDMs. Y_EVs induced less pro-inflammatory gene expression, while O_EVs exhibited a more varied impact, promoting both pro- and anti-inflammatory markers. However, neither EV population induced a clearly defined 'M1' or 'M2' macrophage phenotype. We also report that EVs elicited responses that differed markedly from those induced by whole plasma. Plasma from old mice had strong pro-inflammatory effects on Y_BMDMs, increasing Il1b, Nlrp3 and Tnfa. However, O_EVs did not have these effects, supporting current evidence that EVs are a separate component of circulating factors during ageing. More research is needed to elucidate specific factors involved in inflammageing processes.

Conclusions: Our findings reveal age-related differences in EV cargo and function, with young EVs tending to suppress inflammatory markers more effectively than aged EVs. However, this is not straightforward, and EVs often promoted both M1 and M2 markers. These results suggest that EVs are a distinct component of circulating factors and hold potential for therapeutic strategies aimed at mitigating age-related inflammation and immune dysregulation.

Keywords: Ageing; Aging; Blood plasma; Cytokine; Exosome; Inflammation; Macrophage; miRNA.

Fig. 1

Comparison of extracellular vesicles isolated from young and old mouse plasma (a) Schematic diagram of project experimental design. Y_EV = young mouse plasma extracellular vesicle, O_EV = old mouse plasma extracellular vesicle, Y_BMDM = young mouse bone marrow-derived macrophage, O_BMDM = old mouse bone marrow derived-macrophage. The table shows the factorial experimental design. (b) Mean diameter of Y_EV and O_EVs, determined by NTA. (c) Mode diameter of Y_EVs and O_EVs, determined by NTA. (d) Total particle concentration of Y_EVs and O_EVs, determined by NTA. (e) High magnification and low magnification cryoEM images of Y_EV and O_EV isolates, with representative NTA peaks. EVs are indicated by arrows in the low magnification image. (f) Total protein concentrations of Y_EV and O_EV isolates. (g) Particle: protein ratio for Y_EV and O_EV isolates. (h) Total protein concentration of young (Y) and old (O) mice plasma. Each data point represents EVs isolated from a separate animal. Bar heights represent the mean and error bars show standard error of the mean. Y_EVs were compared to O_EVs by unpaired two-way t-test. ns = not significant (P > 0.05), * = P ≤ 0.05, ** = P < 0.001

Fig. 2

Comparison of bone marrow derived macrophages (BMDMs) isolated from young and old mice (a) Experimental timeline. Bone marrow-derived cells (BMCs) were isolated, differentiated with M-CSF, washed and switched to EV-depleted FBS medium at D6, and analysed at D8. (b) Representative flow cytometry histograms of young (Y_) and old (O_) mouse BMCs at D1 and BMDMs at D8. Positive % for F4/80 (Y axis) and CD11b (X axis) is shown. N ≥ 3 independent batches were analysed. The grey histogram shows isotype control samples. (c) Flow cytometry showing positive population (X axis) for CD206, F4/*), CD11b and CD11c. N = 3 independent batches were analysed. Samples were compared by two-way ANOVA. (d) Quantification of macrophage markers by RT-qPCR in naïve Y_ (n = 9) and O_ BMDMs (n = 3) at D8. Expression was normalised to Hnrnpa1 and Y_BMDMs and O_BMDMs were compared by one-way ANOVA, with Tukey’s post-test, indicated by *. (e) Example images of Y_/O_BMDMs in naïve, IL-4 or LPS-polarised conditions. Data points show samples from separate mice. Bar heights show the mean, error bars show the standard error of the mean. * = P ≤ 0.05, ** = P < 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Fig. 3

Comparison of miRNA cargo isolated from young and old mouse plasma EVs (a) Schematic diagram showing timeline of miRNA isolation, quality control checks and qPCR-based array. Plasma EVs from three mice per group were analysed. (b) Total miRNA yield from Y_EVs and O_EVs. Samples were compared by unpaired t-test. (c) Principal components analysis (PCA) of Y_EV (silver) and O_EV (green) miRNA cargo. (d) Cycle threshold (CT) values for spike-in miRNAs UniSP6 (pre-reverse transcription), UniSP2, 4 and 5 representing high, medium and low expressed miRNAs, and UniSP3 inter-plate calibration (IPC). cel-miR-39-3p is also included. (e) Percentage of miRNAs with high (CT ≤ 30), medium (30-34.9), low (35–40) and absent expression out of 752 assayed miRNAs. No significant differences in populations were detected. (f) The most abundant miRNAs in Y_EV and O_EV samples, listed from highest to lowest. (g) Scatter diagram of Y_EV (x axis) vs. O_EV (Y axis) miRNA expression levels normalised using NormFinder. A correlation value is shown, and some highly abundant miRNAs are annotated. (h) Volcano plot showing statistical significance (Y axis) against fold-change (X axis). The Y axis line represents a P value of 0.05 and X axis lines show fold changes of + 2.0 or -2.0. Significantly different miRNAs are annotated. (j) Venn diagram showing overlap of the 50 most abundant miRNAs in Y_EV and O_EV samples. The miRNAs found in only one population are annotated. Target prediction of differentially-regulated miRNAs relevant to macrophages

Fig. 4

Response of young and old mouse BMDMs to known polarising stimuli (a) Experimental timeline. At D7 BMDMs were stimulated with IL-4 or LPS. At D8 the BMDMs were assessed by qPCR. (b) Log2fc of gene expression changes in Y_BMDMs following treatment with IL-4 (teal colour bars) or LPS (pink bars), compared to naive (PBS-treated) BMDMs which were assigned a log2fc of zero. Significant changes after LPS are shown by an asterisk (*) and changes with IL-4 are shown by hash (#). Samples were compared to naïve cells by 2-way ANOVA with Dunnett’s multiple comparison correction. (c) Log2fc of gene expression changes in O_BMDMs following treatment with IL-4 or LPS. (d) Volcano plot showing comparison between O_ and Y_BMDMs treated with IL-4. The X axis shows log2-fold changes and the Y axis shows P values (-log10 transformed to enable fitting to the graph). The dotted line on the Y axis indicates p = 0.05 and those above the line are considered significant. O_BMDM and Y_BMDM results were compared by unpaired t-test with Holm-Sidak correction for multiple comparisons. (e) Volcano plot showing comparison between O_ and Y_BMDMs treated with LPS. Each data point shows a separate BMDM sample, bar heights show the mean, and error bars show the standard error of the mean. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Fig. 5

Response of young and old BMDMs to young and old mouse plasma (a) Experimental timeline. At D7 BMDMs were stimulated with young mouse plasma (Y_PL) or old mouse plasma (O_PL) for 24 h. (b) Log2fc of gene expression changes in Y_BMDMs following treatment with Y_PL (white bars) or O_PL (green bars), compared to naive (PBS-treated) Y_BMDMs. Significant changes after Y_PL are shown by an asterisk (*) and changes with O_PL are shown by hash (#). Comparisons of Y_PL vs. O_PL are shown by $. (c) Log2fc of O_BMDMs treated with Y_PL or O_PL. (d) Volcano plot showing comparison of O_ and Y_BMDMs treated with Y_PL. (e) Volcano plot showing comparison of O_ and Y_BMDMs treated with O_PL. Data points show separate BMDM samples, bar heights show the mean and error bars show the standard error of the mean. For all comparisons * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Fig. 6

Response of young and old BMDMs to young and old mouse plasma EVs (a) Representative confocal microscope images of EV internalisation by BMDMs. The example shows Y_BMDMs after 30 min incubation with AF680-labelled Y_EVs. The right column shows the two annotated inset areas at higher magnification. 10 μm scale bars are shown. (b) Experimental timeline of EV-induced BMDM polarisation experiment. (c) Log2fc of gene expression changes in Y_BMDMs following treatment with Y_EVs (white bars) or O_EVs (green bars), compared to naive (PBS-treated) BMDMs. Significant changes after Y_EVs are shown by a hash (#) and changes with O_EVs are shown by an asterisk (*). Comparisons of Y_EVs vs. O_EVs are shown by $. (d) Log2fc of gene expression changes in O_BMDMs following treatment with Y_EVs or O_EVs. (e) Volcano plot showing comparison between O_ and Y_BMDMs treated with Y_EVs. The Y axis shows -log10-transformed P values (to enable plotting) and the X axis shows log2 fold change. The dotted line on the Y axis indicates p = 0.05 and those above the line are considered significant. (f) Volcano plot showing comparison between O_ and Y_BMDMs treated with O_EVs. Bar heights show the mean and error bars show the standard error of the mean. Data points show separate BMDMs samples. For all comparisons * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001

Fig. 7

Hierarchical clustering of BMDM gene expression across all experimental conditions (a) Heatmap showing Z-scores of normalised (mRNA/Hnrnpa1) gene expression in naïve and treated Y_BMDMs. Hierarchical clustering is indicated on the left. b) Heatmap for O_BMDMs. Both heatmaps are presented using the same colour scale, shown in the upper right. Y_EV = young mouse plasma EV, O_EV = old mouse plasma EV, Y_PL = young mouse plasma, O_PL = old mouse plasma