Effects of Mesenchymal Stem Cell-Derived Exosomes on Experimental Autoimmune Uveitis

Abstract

We previously demonstrated that mesenchymal stem cells (MSCs) ameliorated experimental autoimmune uveoretinitis (EAU) in rats. Recently, MSC-derived exosomes (MSC-Exo) were thought to carry functions of MSCs. In this study, we tested the effect of local administration of human MSC-Exo on established EAU in the same species. Rats with EAU induced by immunization with interphotoreceptor retinol-binding protein 1177-1191 peptide were treated by periocular injections of increasing doses of MSC-Exo starting at the disease onset for 7 consecutive days. The in vitro effects of MSC-Exo on immune cell migration and responder T cell proliferation were examined by chemotactic assays and lymphocyte proliferation assays, respectively. We found that MSC-Exo greatly reduced the intensity of ongoing EAU as their parent cells by reducing the infiltration of T cell subsets, and other inflammatory cells, in the eyes. Furthermore, the chemoattractive effects of CCL2 and CCL21 on inflammatory cells were inhibited by MSC-Exo. However, no inhibitory effect of MSC-Exo on IRBP-specific T cell proliferation was observed. These results suggest that MSC-Exo effectively ameliorate EAU by inhibiting the migration of inflammatory cells, indicating a potential novel therapy of MSC-Exo for uveitis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Identification of MSC and MSC-Exo. (A) Immunophenotypes of the cultured hMSCs were examined by flow cytometry. The vast majority of cells (>90%) were positive for CD29, CD90, but few cells (<0.5%) expressed CD34, CD45. (B) The hMSCs were maintained in specific induction media for 3 weeks, and the induced cells were then stained by oil red O to indicate adipocytes (B1), von Kossa for osteocytes (B2). For endothelial cell identification, the cells were firstly incubated with antibodies against vWF (B3), and then stained by secondary antibodies conjugated with Alexa Flour 647. (C) Micrographs of scanning electron microscopy of (C1) MSC-Exo and (C2) Fib-Exo show spheroid shaped vesicles at the diameter of about 40-100 nm. Scale bar = 200 nm. (D) Western blot analysis showed that both MSC-Exo and Fib-Exo express CD63, CD9 and CD81.

Figure 2

Periocular injection of MSC-Exo ameliorated uveitis in rats. (A) Both eyes of the immunized rats were injected periocularly with different doses of MSC-Exo (10, 20, 50, and 100 μg) or with an equal volume of PBS (control) on a daily basis for 7 consecutive days starting from the 9th day post immunization. B: 50 μg FIB-exo or an equal volume of PBS was administrated in the same way as above. Asterisks indicate the significant differences between control (black curve) and exosomes treated (blue curve), Values are expressed as the mean ± SD of six rats (12 eyes) per group. *P < 0.05.

Figure 3

Histological assessment of the retina in EAU. Sections of the retina were stained with hematoxylin and eosin, and evaluated for histological damage on 15 and 20 days post immunization. Representative H&E staining images of PBS (A1 and C1, 15 days; B1 and B2 20 days), MSC-exo (A3, 15 days; A4, 20 days), and FIB-exo (B3, 15 days; B4, 20 days) treated groups are shown. Results are expressed quantitatively as histopathological scores (C and D). Values are expressed as the mean ± SD of six rats (12 eyes) per group. *P 

Figure 4

ERG records of the rats. ERG was carried out in both eyes and Ten responses to 0.01 and 3.0 cd × s/m2 white light flash (10 μs, 0.1 Hz) from a Ganzfeld integrating sphere were amplified and averaged. A wave (A and B) and b wave (C and D) amplitudes of dark adapted ERG at 12, 15 and 20 days post immunization were analysed and compared between MSC-exo treated group and control group. Values are expressed as the mean ± SD of six rats (12 eyes) per group. *P < 0.05.

Figure 5

Analysis of the leukocytes infiltration in eyes. (A and B) On day 15 after immunization, representive figures of FACS analysis of CD4+, Gr-1+, CD161+ and CD68+ cells in eyes; C: CD68 immunostaining was used to observe macrophage infiltration in the eyes on day 15 and 20 after immunization (C1: control goup, day 15; C2: control group, day 20; C3: MSC-exo treated group, day 15; C4: MSC-exo treated group, day 20). Values are expressed as the mean ± SD of six rats (12 eyes) per group. *P < 0.05.

Figure 6

Analysis of T cell subsets from eye and lymph node. For CD4+CD25+Foxp3+ cell detection, cells were first gated on CD4+ cells, and then gated on CD25+ and Foxp3+ cells. (A) Representive figures of intracellular staining of IFN-γ, IL-17 and CD25+Foxp3+ in eyes-derived CD4+T cells (left) and in lymph nodes-dvrived CD4+T cells (right) from Control and MSC-exo treated rats with EAU. (B) the percentage of CD4+IFN-γ+, CD4+IL-17+ and CD4+CD25+Foxp3+ cells in eyes. (C) the percentage of CD4+IFN-γ+, CD4+IL-17+ and CD4+CD25+Foxp3+ cells in cervical draining lymph nodes. Values are expressed as the mean ± SD of six rats (12 eyes) per group. *P < 0.05.

Figure 7

The effect of MSC-exo on the chemotaxis of leukocytes. IRBP-specific spleen monocytes prepared from immunized rat on day 12 post immunization were stimulated for 2 days with R16, and then added to the upper well of a microchemotaxis device and incubated in RPMI 1640 medium containing CCL2 or CCL21 with or without MSC-Exo (10 ug/ml). The cells that migrated to the lower well after 4 h were collected and the number of CD4+ (A), CD161+ (A), CD68+ (B) and Gr+ (B) cells were determined by flow cytometry. *P < 0.05.

Figure 8

The effect of MSC-eso on T cell proliferation. T cells and APCs were prepared on day 12 after immunization, from lymph nodes and spleens of EAU rats, and were incubated (T cells/APCs, 1:1) for 48 hours with graded doses of R16 (0, 1, 10 and 30 μg/ml) and MSC-Exo (0, 0.1, 0.5, 1 and 10 μg/ml) (A), or with ConA (0, 2.5, 5, 10 μg/ml) and MSC-Exo (1 and 10 μg/ml). T cell proliferation was determined by measurement of BrdU incorporation. *P < 0.05.