miR-204-containing exosomes ameliorate GVHD-associated dry eye disease

Abstract

Graft-versus-host disease (GVHD)–associated dry eye disease is characterized by extensive inflammatory destruction in the ocular surface and causes unbearable pain and visual impairment. Current treatments provide limited benefits. Here, we report that exosomes from mesenchymal stromal cells (MSC-exo) administered as eye drops notably alleviate GVHD-associated dry eye disease by suppressing inflammation and improving epithelial recovery in mice and humans. In a prospective clinical trial, 28 eyes with refractory GVHD–dry eye disease exhibited substantial relief after MSC-exo treatment, showing reduced fluorescein scores, longer tear-film breakup time, increased tear secretion, and lower OSDI scores. Mechanistically, MSC-exo reprogramed proinflammatory M1 macrophages toward the immunosuppressive M2 via miR-204–mediated targeting of the IL-6/IL-6R/Stat3 pathway. Blockade of miR-204 abolished the effects of MSC-exo, while overloading L929-exo with miR-204 markedly attenuated dry eye. Thus, this study suggests that MSC-exo are efficacious in treating GVHD-associated dry eye disease and highlights miR-204 as a potential therapeutic agent.

Fig. 1.. Topical administration of MSC-exo alleviated dry eye symptoms in BAC-stimulated mice.

(A) Representative images of slit lamp and fluorescein staining showing severe corneal edema and obvious epithelial defects (detected by fluorescein staining) in the eyes of BAC-stimulated mice treated with L929-exo or PBS. In contrast, BAC-stimulated mice treated with MSC-exo eye drops presented with transparent cornea and reduced fluorescein staining areas, similar to the PBS-control normal mouse eye. Unexpectedly, mice treated with artificial tears did not show similar reduction of corneal edema and epithelial defects. (B) Fluorescein staining scores of BAC-stimulated mice after treatment with MSC-exo or L929-exo. n = 12 mice, one-way ANOVA and Tukey’s post hoc test. (C) Cotton thread test showed substantially increased tear volume in BAC-stimulated mice treated with MSC-exo compared to PBS-, L929-exo–, or artificial tear–treated BAC mice. n = 12 mice, one-way ANOVA and Tukey’s post hoc test. n.s., not significant. **P < 0.01 and ***P < 0.001.

Fig. 2.. The GVHD-associated dry eye disease was recovered by MSC-exo eye drops.

(A) Slit lamp and fluorescein staining showing the representative ocular changes of hPBMC-induced GVHD in mice. The extensive epithelial defects were observed in the eyes of GVHD mice treated with L929-exo, while the MSC-exo eye drop group presented with reduced fluorescein staining areas. (B) The equal fluorescein staining scores in the experiment groups before any treatments indicate the baseline. After MSC-exo treatment, the score notably decreased, while there were no statistical differences between artificial tears and L929-exo. n = 8 mice, one-way ANOVA and Tukey’s post hoc test. (C) MSC-exo, but not artificial tear treatment, could increase tear volume. n = 8 mice, one-way ANOVA and Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.. MSC-exo suppressed dry eye symptoms in patients with GVHD-associated dry eye disease.

(A) Representative images of ocular anterior segment under slit lamp and fluorescein examination before and 14 days after MSC-exo treatment. All 28 enrolled eyes with refractory GVHD–associated dry eye disease had no response to topical steroid, artificial tears, or even autologous serum. After MSC-exo eye drop usage, dry eye symptoms and pink eye were alleviated, and the cornea was more transparent with less punctate erosion areas, indicating that MSC-exo can exert a potent antixerophthalmic effect in humans. (B) The relevant information and symptom descriptions of the enrolled patients. (C) Fluorescein scores of individual eye with MSC-exo treatment at D0 and D14 showed decreased trend of fluorescein score in all eyes. (D) After 14-day usage of MSC-exo eye drops, more than half of the enrolled eyes presented with increased tear volume by Schirmer’s test. (E) More stable tear film was observed after MSC-exo treatment with increased tear-film breakup time. (F) Except for two patients, the others reported amelioration of dryness symptoms and improved quality of life, as reflected by decreased OSDI scores after MSC-exo treatment. (G) MSC-exo treatment played no obvious impacts on intraocular pressure. (C to G) n = 28 eyes (14 patients), paired Student’s t tests. *P < 0.05 and ***P < 0.001.

Fig. 4.. MSC-exo could suppress the corneal inflammation during dry eye.

(A) Corneal sections by hematoxylin and eosin staining revealed substantial loss of epithelium in the PBS and L929-exo treatment groups in BAC model, with reduced thicknesses of the total central cornea and the epithelium layer. After MSC-exo treatment, the BAC-stimulated cornea exhibited a regular and well-organized corneal structure, with corneal thickness recovery. n = 6 eyes, one-way ANOVA and Tukey’s post hoc test. Scale bar, 50 μm. (B) Immunofluorescence of corneal section revealed decreased TUNEL⁺ corneal cells, increased Ki-67⁺ proliferative cells, and less CD11b⁺ macrophage infiltration in the MSC-exo group in comparison to the PBS or L929-exo treatment group. n = 3 eyes, one-way ANOVA and Tukey’s post hoc test. Scale bar, 50 μm. (C) In comparison to the L929-exo group, the MSC-exo group displayed reduced levels of proinflammatory cytokines, including Il-6, Il-1β, and Il-17a. n = 6 eyes, unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.. Macrophages were reprogrammed by MSC-exo from M1 to M2 phenotype in vivo and in vitro.

(A) CD9⁺ exosomes were isolated from MSCs transfected with CD9-Tdtomato vector and administered as eye drops to BAC-stimulated mice. Immunochemical analysis of corneal whole mounts detected the colocalization of CD9-Tdtomato-MSC-exo with CD11b⁺ macrophages, suggesting that macrophages were targets of MCS-exo in the cornea. Scale bar, 10 μm. (B) Detection of reduced numbers of CD68⁺CD11b⁺ activated macrophages in MSC-exo–treated corneal flat mounts. Scale bar, 50 μm. (C) Representative cytometry plots and graphs of CD11b⁺ macrophages in the cornea of BAC-stimulated mice. Quadrants show percent CD11c-, CD86-, CD206-, and/or CD11b-expressing cells. n = 12 eyes (four corneas mixed in one sample), one-way ANOVA and Tukey’s post hoc test. (D) The immunofluorescence of M1 and M2 marker (CD86 and CD206, respectively) on cultured raw264.7 macrophages. Scale bar, 50 μm. n = 3, one-way ANOVA and Tukey’s post hoc test. (E) Western blot shows decreased expression of CD86, with increased Arg1 and CD206 expression after MSC-exo treatment in H₂O₂-stimulated raw264.7 cells. n = 3, one-way ANOVA and Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 6.. miR-204 was required for the antixerophthalmic effect of MSC-exo.

(A) miRNA profiling assays were performed on L929-exo and MSC-exo and GO analysis of the target genes of differentially expressed miRNAs in various processes. (B) Differentially up-regulated miRs in MSC-exo that target genes involved in the regulation of immune responses. (C) Volcano plot of differentially expressed miRNAs in MSC-exo than L929-exo. Red dots, up-regulated miRNAs; blue dots, down-regulated miRNAs with a >2.0-fold change. (D) qPCR analysis of miR-204-5p expression in the cornea tissue after MSC-exo treatment. n = 6 eyes, one-way ANOVA and Tukey’s post hoc test. (E) Slit lamp and fluorescein staining analysis of BAC-stimulated mice treated with PBS or exosomes indicated in the figure. Fluorescein staining scores are indicated in the right. L929-miR-204-exo showed similar therapeutic potential with the MSC-exo group. n = 10 mice, one-way ANOVA and Tukey’s post hoc test. ***P < 0.001.

Fig. 7.. miR-204 was the candidate mediator of MSC-exo for macrophage reprogramming from M1 to M2 by targeting IL-6R signaling.

(A) L929-miR-204-exo and MSC-exo treatments suppressed the CD86⁺ M1 macrophages and promoted CD206⁺ M2 ones in the BAC-induced corneal whole-mount staining. Scale bar, 50 μm. (B) Flow cytometry showed that, in the BAC-induced dry eye corneas, the percentage of CD11b⁺CD11c⁻ macrophages decreased after L929-miR-204-exo or MSC-exo treatment compared with L929-miR-NC-exo controls, with more prominent reduction in the MSC-exo group. In addition, both treatments inhibited the percentage of CD11b⁺CD11c⁻CD86⁺ M1 macrophages to a similar extent. n = 12 eyes (four corneas mixed in one sample), one-way ANOVA and Tukey’s post hoc test. (C) mRNA expressions of IL-6, IL-1β, and CD86 were decreased after L929-miR-204-exo and MSC-exo treatments in comparison to the L929-miR-NC-exo group. n = 6 eyes, one-way ANOVA and Tukey’s post hoc test. (D) Predicted regulatory networks of genes targeted by miR-204. (E) Luciferase reporter assay demonstrating direct interaction of miR-204-5p with 3′UTR of the Il-6r gene. One-way ANOVA and Tukey’s post hoc test. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Western blot analysis showing the suppression of IL-6Rα, IL-6, and p-Stat3 by mmu-miR-204-5p precursor.

Fig. 8.. Schematic drawing showing that MSC-exo eye drops alleviate GVHD-associated dry eye disease by reprogramming M1 macrophages to M2 via miR-204–mediated targeting of IL-6R/Stat3 pathway.

The miR-204 in MSC-exo directly targeted the Il-6r gene and suppressed the activation of IL-6R/Stat3 pathway, resulting in the M1 macrophages in the dry eye cornea to reprogram toward immunosuppressive M2 phenotype. MSC-exo, which are enriched in miR-204, could restore the ocular surface homeostasis by switching M1 to M2 and ameliorate the inflammatory destruction during the GVHD-associated dry eye disease.