MSC-Derived Small Extracellular Vesicles Attenuate Autoimmune Dacryoadenitis by Promoting M2 Macrophage Polarization and Inducing Tregs via miR-100-5p

Abstract

Background: Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been increasingly proved as promising immunomodulators against some autoimmune disorders. However, the possible effect and the underlying mechanism of MSC-sEVs in autoimmune dry eye have been rarely studied.

Methods: Small extracellular vesicles from human umbilical cord mesenchymal stem cells (hUC-MSC-sEVs) were subconjunctivally injected to rabbit dry eye model, and their preventive or therapeutical effects were assessed by recording the clinical and histological scores. Quantitative real-time PCR (Q-PCR), western blot and flow cytometry were performed to evaluate the immunomodulatory effects of hUC-MSC-sEVs on macrophages and T regulatory cells (Tregs) both in vivo and in vitro, and the in vitro T cell proliferation was detected by Bromodeoxyuridine (BrdU) assay. In addition, high expression of miR-100-5p in hUC-MSC-sEVs was identified by Q-PCR, and the functional role of sEVs-miR-100-5p on macrophages was explored by a series of co-culture experiments using sEVs derived from hUC-MSCs transfected with miR-100-5p inhibitor.

Results: We firstly demonstrated that hUC-MSC-sEVs had the preventive and therapeutical effects on rabbit autoimmune dacryoadenitis, an animal model of Sjögren's syndrome (SS) dry eye. Further investigation revealed that hUC-MSC-sEVs administration effectively elicited macrophages into an anti-inflammatory M2 phenotype and elevated the proportion of Tregs both in vivo and in vitro, which contributed to reduced inflammation and improved tissue damage. Importantly, hUC-MSC-sEVs-educated macrophages with M2-like phenotype exhibited strong capacity to inhibit CD4+ T cell proliferation and promote Treg generation in vitro. Mechanistically, miR-100-5p was highly enriched in hUC-MSC-sEVs, and knockdown of miR-100-5p in hUC-MSC-sEVs partially blunted the promotion of hUC-MSC-sEVs on M2 macrophage polarization and even attenuated the effect of hUC-MSC-sEVs-educated macrophages on T cell suppression and Treg expansion.

Conclusion: Our data indicated that hUC-MSC-sEVs alleviated autoimmune dacryoadenitis by promoting M2 macrophage polarization and Treg generation possibly through shuttling miR-100-5p. This study sheds new light on the application of MSC-sEVs as a promising therapeutic method for SS dry eye.

Keywords: autoimmune dacryoadenitis; human umbilical cord mesenchymal stem cells; macrophages; miR-100-5p; small extracellular vesicles; tregs.

Figure 1

Characterization of hUC-MSCs and hUC-MSC-sEVs. (A) Flow cytometry analysis for the immunophenotypic surface markers (CD29, CD44, CD105, CD73, CD90, CD45, CD34, CD11b and HLA-DR) of hUC-MSCs. Blue histograms represent the isotype controls, and the red solid peak represents the marker indicated. (B) Representative images of osteogenic (alizarin red S staining), adipogenic (Oil Red O staining) and chondrogenic (Alcian Blue staining) differentiation assay. (C) Representative transmission electron micrograph of hUC-MSC-sEVs. (D) Nanoparticle tracking analysis for measurement of hUC-MSC-sEVs size. (E) Western blotting of marker proteins (CD81, CD9, CD63, TSG101 and Calnexin) in hUC-MSC-sEVs and hUC-MSCs.

Figure 2

The preventive effects of hUC-MSC-sEVs on rabbit autoimmune dacryoadenitis. (A) Schematic diagram of hUC-MSC-sEVs administered at the early stage of rabbit autoimmune dacryoadenitis. (B, C) Representative images and scores of fluorescein staining in cornea. (D) The mean values of tear break-up time. (E) Quantification of tear production by Schirmer’s test. (F) Quantification of the numbers of lymphocytic foci per 4mm² in LGs. (G) Representative photographs of H&E staining in LGs and conjunctivas. Arrows indicate infiltrating lymphocytes. n = 9 rabbits per group (C–F). Data were shown as mean± SD. * hUC-MSC-sEVs group versus untreated group, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

hUC-MSC-sEVs administration promoted M2 macrophage polarization and induced Treg generation in vivo. LGs were collected from rabbits in the untreated and hUC-MSC-sEVs group at week 8. (A) Representative immunofluorescent histological images of CD68 (pan macrophage marker) expression in LGs, and quantification of percent area of CD68+ cells. (B) Gene expression of M1 macrophage markers (NOS2, IRF5, TNF-α, IL-1β and IL-6) and M2 macrophage markers (Arg1, CD206, KLF4, IL-10 and TGF-β) in LGs. (C) Representative western blot images and quantification of relative band intensities for NOS2 and Arg1 in LGs. (D) Gene expression of Foxp3 and Nurr1 in LGs. (E) Flow cytometric analysis of the percentage of CD4+Foxp3+T cells among lymphocytes isolated from LGs. Representative data from at least three independent experiments were presented as mean± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

The therapeutic effects of hUC-MSC-sEVs on rabbit autoimmune dacryoadenitis. (A) Schematic representation of hUC-MSC-sEVs administered at the developed stage of rabbit autoimmune dacryoadenitis. (B, C) Corneal fluorescein staining images and grading scores. (D) Tear break-up time. (E) Tear production. (F) Numbers of lymphocytic foci per 4 mm² in LGs. (G) Representative specimens of H&E staining in LGs and conjunctivas. Arrows indicate infiltrating lymphocytes. n = 5 rabbits per group (C–F). Data were shown as mean± SD. *P < 0.05.

Figure 5

hUC-MSC-sEVs regulated M2 macrophage polarization and Treg differentiation in vitro. PBMCs isolated from model rabbits were stimulated with irradiated pLGECs, and then were treated with or without hUC-MSC-sEVs (5 μg/ml). (A) Gene expression profiles of M1 markers (NOS2, IRF5, TNF-α, IL-1β and IL-6) and M2 markers (Arg1, CD206, KLF4, IL-10 and TGF-β) in PBMCs. (B) Western blot analysis of NOS2 and Arg1 protein level in PBMCs. (C) The mRNA expression of Foxp3 and Nurr1 in PBMCs. (D) Representative flow cytometry showing the frequency of CD4+Foxp3+ T cells in PBMCs. Data were from at least three independent experiments and presented as mean± SD. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

Figure 6

hUC-MSC-sEVs converted inflammatory macrophages to M2 phenotype, which suppressed T cell proliferation and increased Treg frequency. (A) Representative images of the uptake of PKH-26-labeled hUC-MSC-sEVs (red) by human-monocyte-differentiated macrophages (called as Mac, DAPI blue). (B, C) LPS+IFN-γ-stimulated Mac was co-cultured with or without 5 μg/ml hUC-MSC-sEVs for 48h. (B) Q-PCR analysis of M1 markers (NOS2, IRF5, TNF-α, IL-1β and IL-6) and M2 markers (Arg1, CD206, KLF4, IL-10 and TGF-β). (C) Western blot assay for NOS2 and Arg1 protein level. (D, E) After incubated with hUC-MSC-sEVs, macrophages were collected and cultured together with human CD4+ T cells for 5 days in the presence of anti-CD3/-CD28 antibodies. (D) BrdU assay for CD4+ T cell proliferation. (E) Flow cytometric analysis for CD4+Foxp3+ T cells. sEVs, hUC-MSC-sEVs; LPS+IFN-γ-stim., LPS+ IFN-γ-stimulation. Representative data from at least three independent experiments were presented as mean± SD. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

Figure 7

miR-100-5p transferred by hUC-MSC-sEVs modulated macrophage phenotype and function. (A) Q-PCR analysis of miR-100-5p levels in hUC-MSC-sEVs, 293T-sEVs and HDF-sEVs. (B) Representative images of the internalization of miR-100 inhibitor transfected (green) PKH-26-labeled hUC-MSC-sEVs (red) by THP-1 derived macrophages (DAPI blue). (C–E) LPS+IFN-γ-stimulated Mac was co-cultured with miR-100-5p inhibitor-sEVs or NC inhibitor-sEVs for 48h. (C) Q-PCR analysis of M1 markers (NOS2, IRF5, TNF-α, IL-1β and IL-6) and M2 markers (Arg1, CD206, KLF4, IL-10 and TGF-β). (D, E) Protein analysis for NOS2 and Arg1. (F, G) THP-1 derived macrophages pretreated with miR-100-5p inhibitor-sEVs or NC inhibitor-sEVs were co-cultured with human CD4+ T cells stimulated by anti-CD3/-CD28. (F) BrdU assay for CD4+ T cell proliferation. (G) Flow cytometry for CD4+CD25+Foxp3+ T cells. sEVs, hUC-MSC-sEVs; LPS+IFN-γ-stim., LPS+IFN-γ-stimulation. NC, negative-control. Representative data from at least three independent experiments were presented as mean± SD. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.