Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis

Abstract

Objectives: Mesenchymal stem cells (MSCs) release extracellular vesicles (EVs) that display a therapeutic effect in inflammatory disease models. Although MSCs can prevent arthritis, the role of MSCs-derived EVs has never been reported in rheumatoid arthritis. This prompted us to compare the function of exosomes (Exos) and microparticles (MPs) isolated from MSCs and investigate their immunomodulatory function in arthritis. Methods: MSCs-derived Exos and MPs were isolated by differential ultracentrifugation. Immunosuppressive effects of MPs or Exos were investigated on T and B lymphocytes in vitro and in the Delayed-Type Hypersensitivity (DTH) and Collagen-Induced Arthritis (CIA) models. Results: Exos and MPs from MSCs inhibited T lymphocyte proliferation in a dose-dependent manner and decreased the percentage of CD4⁺ and CD8⁺ T cell subsets. Interestingly, Exos increased Treg cell populations while parental MSCs did not. Conversely, plasmablast differentiation was reduced to a similar extent by MSCs, Exos or MPs. IFN-γ priming of MSCs before vesicles isolation did not influence the immunomodulatory function of isolated Exos or MPs. In DTH, we observed a dose-dependent anti-inflammatory effect of MPs and Exos, while in the CIA model, Exos efficiently decreased clinical signs of inflammation. The beneficial effect of Exos was associated with fewer plasmablasts and more Breg-like cells in lymph nodes. Conclusions: Both MSCs-derived MPs and Exos exerted an anti-inflammatory role on T and B lymphocytes independently of MSCs priming. However, Exos were more efficient in suppressing inflammation in vivo. Our work is the first demonstration of the therapeutic potential of MSCs-derived EVs in inflammatory arthritis.

Keywords: cell therapy; extracellular vesicles; mesenchymal stem cells; rheumatoid arthritis; trophic factors.

Figure 1

Freshly isolated extracellular vesicles from murine MSCs exert immunosuppressive functions. (A) Experimental protocol for isolation of total extracellular vesicles (EVs) using differential ultracentrifugation. (B) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with naïve or IFN-γ (20 ng/mL)-primed MSCs (n=3 biological replicates). (C) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (C) or incubated with ultracentrifuged production medium (UM) or naïve MSCs or MSCs-conditioned medium (CM) pre (Pr)- or post (Po)-100,000 × g centrifugation according to (A). CM was depleted in cells and debris (by 300 × g and 2500 × g centrifugation steps). MSCs were naïve or primed with 20 ng/mL IFN-γ (n=3 biological replicates). (D) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with increasing amounts of EVs (n=4 biological replicates). (E) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with 50 ng of freshly isolated or freeze-thawed EVs (n=4 biological replicates). (F) TEM analysis of freshly isolated (4°C) or freeze-thawed EVs (-80°C). Bar is 100 nm. (G) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of CD81 exosomal marker on freshly isolated (4°C) or freeze-thawed EVs (-80°C) analyzed by flow cytometry. (H) Number and median size of freshly isolated (4°C) or freeze-thawed EVs (-80°C) by Nano Tracking Analysis. Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test (B, C, D, E) or a Mann-Whitney test (H). *: p<0.05.

Figure 2

Isolation and characterization of exosomes and microparticles isolated from murine MSCs. (A) Experimental protocol for isolation of microparticles (MPs) and exosomes (Exos) from MSCs-conditioned medium using differential ultracentrifugation. (B) Quantification of EVs produced by 10⁶ MSCs and expressed as equivalent protein. MSCs were naïve or primed with 20 ng/mL IFN-γ (n=5 biological replicates). (C) Representative pictures of MPs (18K) and Exos (100 k) by transmission electron microscopy. Bars represent 200 nm in large pictures and inserts for MPs; for Exos, bars are 200 nm for large pictures and 100 nm for inserts. (D) Number and size of Exos (left) and MPs (right) detected in 1 mL (corresponding to 1 µg EVs equivalent protein) by Nano Tracking Analysis. Letters (A to E) indicate various population peaks (n=3 biological replicates). (E) Quantification of Exos and MPs particle numbers related to the quantity of protein (n=3 biological replicates). (F) Mean size of Exos (left) and MPs (right) in the fractions represented in (D) (n=3 biological replicates). (G) Percentage of MPs in each fraction (A to E) related to total MPs (n=3 biological replicates). (H) Expression of MSCs membrane markers (Sca-1, CD44, CD29) and of exosomal markers (CD9, CD81) on Exos (top) and MPs (bottom) isolated from naïve MSCs analyzed by flow cytometry.

Figure 3

MPs and Exos exert immunosuppressive functions on T lymphocyte subsets. (A) Proliferation of Concanavalin A-activated murine splenocytes cultured alone for 3 days (Ctrl) or incubated with increasing amounts (ng) of Exos (left) or MPs (right) from naïve or IFN-γ primed MSCs (n=5 biological replicates). (B) Percentage of CD8⁺IFN-γ⁺ cytotoxic T cells, CD4⁺IFN-γ⁺ Th1 cells, CD4⁺IL-10⁺ Tr1 cells and CD4⁺CD25⁺Foxp3⁺ Treg cells in splenocytes (n=4) when incubated alone or with naïve MSCs and 25 ng of Exos or MPs. (C) Proliferation of Concanavalin A-activated sorted CD4⁺ (left) and CD8⁺ (right) T lymphocytes cultured alone for 3 days (Ctrl) or incubated with naïve MSCs and 50 ng of Exos or MPs from naïve or IFN-γ primed MSCs (n=3 biological replicates). Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test. *: p<0.05 compared to Ctrl.

Figure 4

MPs and Exos exert immunosuppressive functions on B lymphocytes. (A) Percentage of CD138⁺ plasmablasts obtained after activation (Ctrl) or culturing with naïve MSCs or 50 ng Exos or MPs from naïve or IFN-γ primed MSCs (n=5 biological replicates). Representative flow cytometry pictures are shown below. (B) Concentration of total IgG, TNFα, IL6, IL10 in supernatants from plasmablasts in (A) as expressed in arbitrary unit (a.u.) or pg/mL (n=5 biological replicates). (C) Amounts of TGF-β1, PGE2, IL-6, IL1-RA in 1 µg of Exos or MPs as evaluated by ELISA (n=5 biological replicates). Statistical analysis used a non-parametric Kruskal-Wallis test with Dunn's multiple comparison post-test. *: p<0.05compared to Ctrl.

Figure 5

MPs and Exos suppress inflammation in the DTH and CIA models. (A) Inhibition of inflammation in the Delayed-Type Hypersensitivity (DTH) model with increasing doses of MPs or Exos from IFN-γ primed MSCs, as measured by swelling of hind paws at day 6 (n=7 biological replicates). (B) Incidence of mice with inflammation in the collagen-induced arthritis (CIA) model until day 37 at euthanasia (n=15 biological replicates). (C) Inhibition of inflammation as measured by clinical score in the same mice as in (B). (D) Representative 3D reconstruction images of hind paws by µCT analysis showing bone degradation in feet (top) or ankles (bottom) from a Ctrl mouse (left), MPs-treated mouse (middle) and Exos-treated mouse (right). (E) Mean thickness of cuneiform bone from Ctrl mice or mice treated with MPs or Exos as evaluated by histomorphometric µCT analysis. (F) Mean bone degradation of cuneiform bone as evaluated by area/volume parameter by µCT. Statistical analysis used a nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (A, E, F) or a two-way ANOVA followed by Tukey's multiple comparison test (C). *: p<0.05 or **: p<0.01 compared to Ctrl.

Figure 6

Exos are efficient immunosuppressive agents in CIA by decreasing plasmablast differentiation and generating Breg cells. (A) Effect of 250 ng Exos or 600 ng MPs on inflammation in the CIA model as measured by hind paw swelling, fore paw score and global clinical score until day 30 at euthanasia. (B) Determination of the percentage of CD4⁺IFNγ⁺ Th1 cells, CD4⁺IL17⁺ Th17 cells, CD4⁺Foxp3⁺ Treg cells, CD4⁺IL10⁺ Tr1 cells, CD138⁺ plasmablasts and CD19⁺IL10⁺ Breg cells in lymph nodes from control CIA mice and mice treated with Exo or MPs. (C) Quantification of IL-6, TNF-α, IL-1β and IL-10 in supernatants from resting lymph nodes (NA), activated with specific type II collagen (bC2) or ConA. (D) Type II collagen-specific IgG2a/IgG1 antibody ratios in sera of CIA Ctrl mice or mice treated with Exos or MPs at the indicated time points during CIA. Statistical analysis used a two-way ANOVA followed by Tukey's multiple comparison test (A) or a nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (B, C, D) with n=15 biological replicates and *: p<0.05; ***: p<0.001 or ****: p<0.00001 compared to CIA Ctrl.