Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun 2;7(23):eabe0083.
doi: 10.1126/sciadv.abe0083. Print 2021 Jun.

Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis

Dong Gil You  1 , Gyeong Taek Lim  1 , Seunglee Kwon  1 , Wooram Um  1 , Byeong Hoon Oh  1 , Seok Ho Song  1 , Jungmi Lee  1 , Dong-Gyu Jo  2   3   4 , Yong Woo Cho  4   5 , Jae Hyung Park  6   2   4
Affiliations

Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis

Dong Gil You et al. Sci Adv. .

Abstract

Despite the remarkable advances in therapeutics for rheumatoid arthritis (RA), a large number of patients still lack effective countermeasures. Recently, the reprogramming of macrophages to an immunoregulatory phenotype has emerged as a promising therapeutic strategy for RA. Here, we report metabolically engineered exosomes that have been surface-modified for the targeted reprogramming of macrophages. Qualified exosomes were readily harvested from metabolically engineered stem cells by tangential flow filtration at a high yield while maintaining their innate immunomodulatory components. When systemically administered into mice with collagen-induced arthritis, these exosomes effectively accumulated in the inflamed joints, inducing a cascade of anti-inflammatory events via macrophage phenotype regulation. The level of therapeutic efficacy obtained with bare exosomes was achievable with the engineered exosomes of 10 times less dose. On the basis of the boosted nature to reprogram the synovial microenvironment, the engineered exosomes display considerable potential to be developed as a next-generation drug for RA.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Schematic illustration of DS-EXOs as a cell-free therapeutic system for RA.
(A) The surface engineering process of MSC-EXOs by the MGE-mediated click chemistry of ADSCs. This methodology facilitated the introduction of a targeting moiety onto the MSC-EXOs without causing their dysfunction. (B) Mechanism by which the DS-EXOs reprogram macrophages. After systemic administration, the DS-EXOs reach joints inflamed by RA owing to their ability to target activated macrophages. Macrophage reprogramming in RA can be effectively promoted by the targeted delivery of the DS-EXOs. MVB, multivesicular bodies.
Fig. 2
Fig. 2. Biogenesis of DS-EXOs based on the MGE-mediated click chemistry of ADSCs.
(A) Confocal microscopic images of DS-modified ADSCs at different concentrations of Ac4ManNAz. For the MGE-mediated click chemistry, ADSCs were incubated with Ac4ManNAz for 48 hours to generate azide groups on the surface and then treated with serum-free medium containing Cy5.5-labeled DBCO-DS (10 μM) for 2 hours. (B) Quantification of the DS on the ADSCs at different concentrations of Ac4ManNAz. Modified ADSCs were analyzed by flow cytometry to quantify the azide surface expression. (C) Intracellular tracking of DS in MDA-MB-231-CD63-GFP cells. The CD63-GFP cell line was used for MGE-mediated click chemistry to confirm the intracellular colocalization of CD63 and DS during biogenesis. Confocal microscopy images show CD63-GFP (green) and Cy5.5-labeled DS (red) in the MDA-MB-231-CD63-GFP cells. (D) Intracellular tracking of DS in the ADSCs. Time-dependent fluorescence images of the early endosomes and DS were obtained after the MGE-mediated click chemistry of ADSC. Confocal microscopy images show EEA1 (green) and Cy5.5-labeled DS (red) in the ADSCs. (E) Kinetic quantification of confocal imaging in (D). a.u., arbitrary units. (F) Secretion of the DS-decorated extracellular vesicles from the ADSCs. To visualize the extracellular secretion of the DS-decorated extracellular vesicles, the ADSCs were seeded onto a High Grid-500 glass-bottom μ-Dish. After the MGE-mediated click chemistry, images were separately obtained for the same spot using SEM and confocal microscopy. The dotted region in the top panel is magnified in the bottom panel.
Fig. 3
Fig. 3. Physicochemical characteristics of the DS-EXOs.
(A) Schematic illustration of the TFF-based isolation of the DS-EXOs. (B) Production yield of the bare EXOs and DS-EXOs. After surface engineering the ADSCs, the cells were incubated for 24 hours with FBS-free medium, and the DS-EXOs were isolated using TFF. The EXO number per cell was measured using an NTA. Error bars represent the SD (n = 3). NS, not significant. (C) Size distribution of the bare EXOs and DS-EXOs. The hydrodynamic size of the EXOs was measured using an NTA. (D) TEM images of the bare EXOs and DS-EXOs. Samples were dropped on a 200-mesh carbon film–coated grid and negatively stained with uranyl acetate. (E) Confocal microscopy images of exosomal surface markers on the bare EXOs and DS-EXOs. To analyze the exosomal markers, the EXOs were incubated with magnetic microbeads coated with antibody. Confocal microscopy images show anti-CD9 or anti-CD63 (green) and the microbeads. (F) Flow cytometry analysis of the exosomal surface markers on the bare EXOs and DS-EXOs.
Fig. 4
Fig. 4. miRNA profiling of the bare EXOs and DS-EXOs.
(A) Heatmap analysis of the top 50 significantly expressed miRNAs in the bare EXOs and DS-EXOs. Purified total RNA was used for the small RNA sequencing of the EXOs. (B) Schematic representation of the predicted target genes of the top four significantly expressed miRNAs enriched in the bare EXOs and DS-EXOs. The gene clusters (blue) and networks of the miRNAs were visualized using Cytoscape. (C) GO and KEGG pathway analyses of the target genes of the top four significantly expressed miRNAs in the bare EXOs and DS-EXOs. The GO terms and KEGG pathway terms enriched in the predicted target genes of the miRNAs were analyzed using DAVID Bioinformatics. (D) Volcano plot of the miRNAs in the bare EXOs and DS-EXOs. The red dots indicate the differentially expressed miRNAs between the bare EXOs and DS-EXOs. (E) Number of differentially up- and down-regulated miRNAs by fold change and P value. BP, biological processes; CC, cellular components; MF, molecular functions.
Fig. 5
Fig. 5. Targeted delivery of DS-EXOs into the activated macrophages.
(A) Cellular uptake behavior of the Cy5.5-labeled bare EXOs and DS-EXOs in RAW264.7 cells (LPS+ and IFN-γ+). (B) Cellular uptake behaviors of the bare EXOs and DS-EXOs in BMDMs (LPS+ and IFN-γ+). Confocal microscopy images show nuclei (blue) and Cy5.5-labeled EXOs (red) in the cells (A and B). (C) In vivo fluorescence images of the Cy5.5-labeled bare EXOs and DS-EXOs in the CIA mice. The images were obtained using an IVIS Lumina III In Vivo Imaging System or SPECTRAL Lago X with an embedded x-ray. (D) Fluorescence intensity of the bare EXOs and DS-EXOs in the inflamed joints of the CIA mice as a function of time. Error bars represent the SE (n = 3). (E) Whole-body fluorescence imaging of bare EXOs and DS-EXOs in the CIA mice. After removing the hair, Cy5.5-EXOs or Cy5.5-DS-EXOs (1 × 108 particles per head) were systemically administered into the CIA mice. (F) Fluorescence intensity of bare EXOs and DS-EXOs in the whole body of the CIA mice as a function of time. Error bars represent the SE (n = 3). (G) Ex vivo organ distribution images of the bare EXOs and DS-EXOs. (H) Quantification of fluorescence intensity in the inflamed joints and major organs of the CIA mice. Error bars represent the SE (n = 3). (I) Distribution of SR-A and EXOs in the inflamed joints of the CIA mice. **P < 0.01, ***P < 0.005, and ****P < 0.001, analyzed by two-way analysis of variance (ANOVA).
Fig. 6
Fig. 6. In vitro macrophage polarization by DS-EXOs.
(A) Schematic illustration for M1-M2 macrophage polarization by DS-EXOs (left). The RAW264.7 cells (LPS+ and IFN-γ+) were incubated with the bare EXOs or DS-EXOs (1 × 107 or 1 × 108 particles per dish) at 37°C for 48 hours. Confocal microscopy images show iNOS (red) and CD206 (green) in cells (right). (B) Flow cytometry of the M1 and M2 macrophage markers in RAW264.7 cells (LPS+ and IFN-γ+). (C) Quantification of the expression levels of CD206 in EXO-treated RAW264.7 cells (LPS+ and IFN-γ+). Error bars represent the SD (n = 3). (D) Schematic illustration for inhibition of M2-M1 macrophage polarization by DS-EXOs (left). Confocal microscopy images show iNOS (red) and CD206 (green) in EXO-treated RAW264.7 cells (IL-4+) in the presence of LPS and IFN-γ (right). The cells were incubated with the bare EXOs or DS-EXOs (1 × 107 or 1 × 108 particles per dish) at 37°C for 48 hours. (E) Schematic illustration for inhibition of M2-M1 macrophage polarization by DS-EXOs (left). Confocal microscopy images show iNOS (red) and CD206 (green) in EXO-treated RAW264.7 cells (LPS+ and IFN-γ+) in the presence of let-7b-5p and miR-24-3p inhibitors (right).
Fig. 7
Fig. 7. Therapeutic efficacy of the DS-EXOs.
(A) Treatment regimen used for the bare EXOs and DS-EXOs. The red circles represent intravenous injections of the samples. iv, intravenous. (B and C) The severity of arthritis was determined via (B) a visual arthritis scoring system and (C) the paw thickness of the CIA mice. Error bars represent the SE (WT, n = 5; CIA, n = 10). (D) T scores of the inflamed joints in the CIA mice. On day 35 after the first immunization, the T scores of the joints were obtained. Error bars represent the SE (n = 5). (E) Representative images of the CIA mice treated with PBS, bare EXOs (1 × 108 particles per head), DS-EXOs (1 × 107 particles per head), and DS-EXOs (1 × 108 particles per head). (F) Hematoxylin and eosin–stained images of the inflamed joints in each treatment group. (G to I) Histological scores of neutrophil infiltration, synovial inflammation, and cartilage erosion. The histological scores were obtained from the images in (F). Error bars represent the SD (n = 5). (J) Regulation of macrophage heterogeneity by DS-EXOs. For immunohistochemistry, the joints were decalcified and the tissue sections were immunostained with anti-iNOS antibody Alexa Fluor 647 and anti-CD206 antibody Alexa Fluor 488. (K) Quantification of cytokines in the blood of the CIA mice. After 35 days of therapeutic monitoring, the collected blood samples were centrifuged to obtain serum. The cytokine levels in the serum samples were analyzed using ELISA. Error bars represent the SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001, analyzed by two-way ANOVA. Photo credit: Dong Gil You, Sungkyunkwan University.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Olumuyiwa-Akeredolu O.-o., Page M. J., Soma P., Pretorius E., Platelets: Emerging facilitators of cellular crosstalk in rheumatoid arthritis. Nat. Rev. Rheumatol. 15, 237–248 (2019). - PubMed
    1. Smolen J. S., Steiner G., Therapeutic strategies for rheumatoid arthritis. Nat. Rev. Drug Discov. 2, 473–488 (2003). - PubMed
    1. Fortune Business Insights (2019); www.fortunebusinessinsights.com/industry-reports/rheumatoid-arthritis-th....
    1. Smolen J. S., Aletaha D., Barton A., Burmester G. R., Emery P., Firestein G. S., Kavanaugh A., McInnes I. B., Solomon D. H., Strand V., Yamamoto K., Rheumatoid arthritis. Nat. Rev. Dis. Primers. 4, 18001 (2018). - PubMed
    1. Udalova I. A., Mantovani A., Feldmann M., Macrophage heterogeneity in the context of rheumatoid arthritis. Nat. Rev. Rheumatol. 12, 472–485 (2016). - PubMed

Publication types