Protective effects of CCR5-overexpressing mesenchymal stem cells on experimental autoimmune uveitis

Background Introduction

Uveitis is an inflammatory eye disease that seriously threatens vision and can lead to complications such as cataracts, glaucoma, vitreous opacities, retinal detachment, and abnormal retinal vasculature. This disease is widespread globally, and one form is autoimmune uveitis (AU), which is the fourth leading cause of severe vision impairment in industrialized countries. This class of diseases operates through various pathological mechanisms and exhibits a multitude of characteristics across different diseases.

To further understand the pathogenesis of uveitis, researchers have traditionally used the experimental autoimmune uveitis (EAU) animal model to study this disease. Since 1988, researchers have induced EAU through the interphotoreceptor retinoid-binding protein (IRBP), and this model shares many pathological similarities with human uveitis and has been widely used for genetic impact studies, revealing pathogenic mechanisms and testing potential treatment plans. However, neither EAU nor other animal models can fully reproduce all features of human uveitis.

Research Source

The research paper was published in the “Journal of Neuroinflammation,” titled “CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis,” written by Yuan et al. in 2024, with authors primarily from Zhongshan Ophthalmic Center of Sun Yat-sen University and Zhongshan Medical School.

Research Content

Research Process

This study carried out a detailed investigation of the classic mouse EAU model using single-cell RNA sequencing (scRNA-seq) and whole RNA sequencing (RNA-seq) technologies, combined with a variety of molecular and cellular methods. The research process included the following steps:

1. Establishment of the EAU mouse model:

   • C57BL/6 mice were immunized with the 651-670 peptide segment of human IRBP, followed by immunosuppression for 15 days to ensure the successful induction of uveitis.
   • Mice were assessed for disease status via fundus imaging, fluorescein fundus angiography (FFA), and optical coherence tomography (OCT).

2. Single-cell transcriptome analysis:

   • On day 14 after disease induction, retinal tissue was collected for single-cell transcriptome sequencing (scRNA-seq) using the 10x Genomics platform, obtaining transcriptome data for 20,448 single cells.
   • Cluster analysis of different cell types was conducted, identifying the primary cell populations in the retina, including microglia, monocytes/macrophages, T/NK cells, various retinal neurons, and vascular endothelial cells.

3. Whole transcriptome analysis:

   • RNA-seq technology was used for whole-gene expression analysis of the retina. Changes in gene expression were assessed through Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA).

4. Study of Müller Glia function:

   • Müller cells were found to potentially act as antigen-presenting cells (APCs) during EAU.
   • Further cluster analysis of Müller cells revealed gene expression profiles of different subgroups.

5. Analysis of immune cell infiltration:

   • An in-depth analysis of immune cells in the EAU mouse retina was conducted, including infiltration of various subtypes of T cells, as well as intercellular signaling analysis.

6. Migration and therapeutic testing of CCR5-overexpressing MSCs:

   • Human CCR5-overexpressing mesenchymal stem cells (MSCs) were produced, evaluating their chemotactic ability towards CCL5 in vitro and in vivo.
   • MSCs were transplanted into EAU mice, assessing their therapeutic effect, especially on the activation of M1-type microglia, T cell and macrophage infiltration.

Research Results

1. Infiltration of various immune cells into the retina of EAU mice:

   • Fundus imaging showed significant vascular leakage, retinal structure disorganization, and invasion of numerous CD45-positive leukocytes and CD11b-positive myeloid cells as the disease progressed.

2. Decrease in retinal neurons:

   • Single-cell transcriptome analysis revealed a significant decrease in the number of retinal neurons, specifically in bipolar cells, cone cells, and horizontal cells.
   • The whole transcriptome analysis showed downregulation of marker gene expression for all types of neural cells in the EAU-affected retina.

3. Activation and antigen-presenting function of Müller Glia:

   • It was found that during EAU, Müller cells expressed major histocompatibility complex class II (MHC II) genes and showed MHC II protein expression, indicating that Müller cells may act as non-professional antigen-presenting cells.

4. Diversity of T cells in the retina:

   • The infiltrating T cells were predominantly Th1 cells, accounting for 19.83% of all immune cells.

5. Significant upregulation of CCL5 expression:

   • The expression of the CCL5 gene was most pronounced in the EAU retina, with an expression level 2,500 times that of the control group.

6. Migration and therapeutic effect of CCR5-overexpressing MSCs:

   • In vitro experiments confirmed that CCR5-overexpressing MSCs showed significant chemotaxis under the influence of CCL5.
   • In vivo experiments indicated that the retinas of mice transplanted with CCR5-overexpressing MSCs showed a substantial reduction in activated microglia and infiltrating T cells and macrophages. Moreover, compared to the control group, the transplantation group exhibited milder retinal structural damage and lower inflammation scores.

7. Downregulation of NLRP3 inflammasome-related genes:

   • Expression of NLRP3 and IL-1β was significantly downregulated in the retinas of the MSC treatment group.

Research Conclusions

This study revealed extensive damage to retinal cells in EAU, revealed the potential antigen-presenting function of Müller cells in EAU, and found that Th1 cells are the main infiltrating cell type. Through the marked upregulation of CCL5 in the retina, this study proposed and validated a strategy of treating EAU by transplanting CCR5-overexpressing MSCs, significantly improving the MSC’s chemotactic ability and therapeutic effect. This provides a new approach for the MSC treatment of EAU with potential clinical application prospects.

Research Highlights

• Innovative single-cell transcriptome analysis technique: For the first time, scRNA-seq technology was used to comprehensively analyze the classic EAU model induced by IRBP.
• Experimental validation of CCR5-overexpressing MSCs: CCR5 overexpression significantly increased the migration and therapeutic effect of MSCs in the retina, which could expand the application prospects of MSCs in treating autoimmune diseases.
• In-depth analysis of the retinal immune environment: Revealed complex changes in retinal cell types and immune cells, providing important data support for understanding the pathogenesis of EAU.

Materials and Methods

The mice used in the study were bred under ethical standards, with C57BL/6 mice used for EAU model induction, and various imaging and molecular biology techniques applied for detection and analysis. In addition, the study used proteomic data analysis and real-time quantitative PCR to verify experimental results such as migration capability and inflammasome expression.

Discussion

This study used single-cell transcriptome and whole transcriptome sequencing technologies to thoroughly analyze the cellular composition and gene expression changes in the retina of the EAU model. It further explored the potential antigen-presenting function of Müller cells in their activated state and demonstrated through CCR5-overexpressing MSC transplantation experiments the significant effects of this strategy in improving the clinical manifestation of EAU.

Future research could further optimize specific methods for MSC transplantation, such as MSC modification methods, optimum timing for transplantation, quantity of injected cells, and frequency of transplantation, to provide a more comprehensive basis for clinical application.