Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance

Abstract

Biologic aging results in a chronic inflammatory condition, termed inflammaging, which establishes a risk for such age-related diseases as neurocardiovascular diseases; therefore, it is of great importance to develop rejuvenation strategies that are able to attenuate inflammaging as a means of intervention for age-related diseases. A promising rejuvenation factor that is present in young blood has been found that can make aged neurons younger; however, the component in the young blood and its mechanism of action are poorly elucidated. We assessed rejuvenation in naturally aged mice with extracellular vesicles (EVs) or exosomes extracted from young murine serum on the basis of different spectrums of microRNAs in these vesicles from young and old sera. We found that EVs extracted from young donor mouse serum, rather than EVs extracted from old donor mouse serum or non-EV supernatant extracted from young donor mouse serum, were able to attenuate inflammaging in old mice. Inflammaging is attributed to multiple factors, one of which is thymic aging-released self-reactive T cell-induced pathology. We found that the attenuation of inflammaging after treatment with EVs from young serum partially contributed to the rejuvenation of thymic aging, which is characterized by partially reversed thymic involution, enhancement of negative selection signals, and reduced autoreactions in the periphery. Our results provide evidence for understanding of the potential rejuvenation factor in the young donor serum, which holds great promise for the development of novel therapeutics to reduce morbidity and mortality caused by age-related inflammatory diseases.-Wang, W., Wang, L., Ruan, L., Oh, J., Dong, X., Zhuge, Q., Su, D.-M. Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance.

Keywords: age-related thymic involution; chronic inflammation; exosomes; rejuvenation; young serum.

Figure 1

Characteristics of serum-extracted EVs. A) Comparison of the sizes of EVs extracted from young murine serum by the ExoQuick reagent pretreated with and without using 0.2-μm filters. B) Morphology of EVs from young murine serum used in this project for rejuvenation of inflammaging, photographed by atomic force microscopy (AFM). C, D) Different miRNA expression profiles in heatmap (C) and quantified summary (D) of EVs from young vs. old murine serum, analyzed by murine miRNA microarray with Mus musculus miRBase version-21 arrays that contained 1900 unique mature miRNA probes (miRNA microarray service via LC Sciences).

Figure 2

EVs extracted from young serum reduced proinflammatory factors in aged mice. A) Proinflammatory cytokines, IL-6 (left) and IL-1β (right), were increased above baseline in aged murine serum, but were significantly decreased after treatment with EVs extracted from young serum (detailed in Materials and Methods) as measured by ELISA; however, changes in IL-6 expression in mice that were treated with EVs from aged serum or young non-EV serum supernatant could not be observed (left, the rightmost 2 striped bars). B) Proinflammatory factor, TNF-α, was increased above baseline in aged murine spleen, but was significantly reduced after treatment with EVs extracted from young serum, as in panel A, measured by real-time RT-PCR. C) Dot plots show a representative flow cytometric gate profile of splenic CD4⁺IFN-γ⁺ T cells from aged mice after costimulation with anti-CD3ε and anti-CD28 (left). The right panel shows a summarized result of reduction of splenic %CD4⁺IFN-γ⁺ T cells from aged mice after treatment with EVs extracted from young serum. P values are shown in each panel.

Figure 3

EVs extracted from young serum reduced inflammaging in the CNS of aged mice. A) Workflow for isolating non-neural mononuclear cells from brains. B) A representative flow cytometric profile of brain mononuclear cells from young mice and aged mice that were treated with PBS or EVs extracted from young serum (detailed in Materials and Methods). Dot plots in the top row show gates of microglia (CD45^−/lowCD11b⁺ population), Mϕs (CD45^highCD11b⁺ population), and potential lymphocytes (CD45⁺CD11b⁻ population). Dot plots in the bottom row show gates of CD3⁺ population from the CD45⁺CD11b⁻ population in which neural cells were excluded. Two vertical dotted lines in panel B (top) divide CD45 staining into CD45⁻, CD45^low, and CD45^high (from left to right) subsets. C) Summarized results of %CD3⁺ cells in the CD45⁺CD11b⁻ population (left), %Mϕs in non-neural cells (CD45^highCD11b⁺ population; middle panel), and MHC-II expression strength [mean fluorescence intensity (MFI)] in microglia (CD45^−/lowCD11b⁺ population) of mouse brains from the 3 treatment groups. These 3 parameters, which represent inflammation in the CNS, were increased in aged mice, but decreased after injection with EVs extracted from young serum. P values are shown in each panel, and each symbol represents an individual animal sample.

Figure 4

EVs extracted from young serum induced partial rejuvenation of the aged thymus. A) A representative image of murine thymuses showing the aged involuted thymus (middle) compared with the young thymus with normal size (left) was partially increased in size after treatment with EVs extracted from young serum (right). B) A summarized result shows thymic mass changes with a similar tendency as that in panel A. C) A summarized result shows thymocyte number changes with a similar tendency as those in panels A, B. D, E) Summarized results of FoxN1 (D) and ΔNp63 (E) expression, with a real-time RT-PCR assay, in murine thymuses of young and aged mice that were treated with PBS and EVs extracted from young serum, respectively, with the same tendencies as those in panels A–C. P values are shown in each panel.

Figure 5

EVs from young serum induced rejuvenation of age-related abnormal microstructure in the aged thymus. A) Representative thymic cryosections with immunofluorescence staining shows the images of K5 (representing the medulla) vs. K8 (representing the cortex) in the top row, and UEA-1 (representing mature mTECs) vs. β-5T (representing progenitor cTECs) in the bottom row. Thymuses were from aged mice that were treated with PBS (for control) and EVs extracted from young serum. Dotted lines indicate corticomedullary borders, and arrows indicate typical β-5T positive cells. Data are representative of 3 biologic replicates in each group with essentially identical results. B) A summarized semiquantified result of K5⁺ mTECs vs. K8⁺ cTECs in thymuses from aged mice that were treated with PBS (for control) and EVs extracted from young serum, respectively. C) Summarized results of UEA-1 (representing mature mTECs) and β-5T (representing progenitor cTECs) in thymuses from aged mice that were treated with PBS (for control) and EVs extracted from young serum, respectively. P values are shown in each panel, each symbol represents 1 scope of thymic tissue under microscope.

Figure 6

EVs extracted from young serum induced an increase in negative selection–associated molecules in the aged thymus. A) Representative immunofluorescence staining images of Aire⁺ TECs (red) in K8⁺ TEC background (green). Original magnification, ×20. Top panels are young mice that were treated with PBS and aged mice treated with PBS and EVs from young serum, whereas bottom panels are aged mice that were treated with EVs from aged serum and young non-EV serum supernatant. Data are representative of 5 mouse thymuses per treatment group with essentially identical results. B) A summarized result shows the percent area of Aire⁺ TECs under K8⁺ counterstaining on the basis of slides in panel A. Each symbol represents 1 thymic tissue slide with ∼9 tissue slides for each mouse, calculated using ImageJ software. C) Real-time RT-PCR results show the expression of Aire mRNA in the aged thymuses. Aire expression was increased in aged mice that were treated with EVs from young serum compared with aged mice that were treated with PBS vehicle. D) Nur77 signaling strength was increased in CD4⁺ thymocytes of aged mice that were treated with EVs from young serum. Left panel shows histogram of Nur77 in CD4⁺ population, and the right panel shows a summary of relative Nur77 mean fluorescent intensity (MFI) in CD4⁺ populations of 3 groups of mice. E) CD5 signaling strength was increased in CD4⁺ thymocytes of the same aged mice as in panel D. Left panel shows histogram of CD5 in CD4⁺ population, and right panel shows a summary of relative CD5 MFI in CD4⁺ populations of 3 groups of mice. P values are shown in each panel, each symbol represents an individual animal sample.

Figure 7

Chronic autoimmune predisposition or features during inflammaging were reduced in aged mice after treatment with EVs extracted from young serum. A) A schematic workflow shows the process of naturally aged mice that were treated with EVs extracted from young serum, followed by isolation of their serum for analysis of ANAs and isolation of their splenic lymphocytes for adoptive transfer into young Rag^−/− mice. Subsequently, inflammatory cell infiltration into a nonlymphoid organ, namely the salivary gland, was analyzed with hematoxylin and eosin (H&E) staining at 8 wk after transfer. B) Concentrations of serum ANAs were increased in aged mice, but significantly reduced after treatment with EVs from young serum (see Materials and Methods). A Student’s t test was used, and P values are shown in each panel. Each symbol represents an individual animal sample. C) Representative H&E staining images of inflammatory cell infiltration in salivary glands of young Rag^−/− mice, which were infused with spleen lymphocytes from young (treated with PBS, left), aged (treated with PBS, middle), and aged (treated with EVs from young serum, right) mice. Blue arrows indicate microvessels, and yellow dotted lines show the foci of infiltrating lymphocytes from microvessels. Numbers in top panels indicate the numbers of pathologic foci observed in 120 tissue slides. Data are representative of 3 biologic replicates in each group with essentially identical results.