High-throughput RNA-sequencing identifies mesenchymal stem cell-induced immunological signature in a rat model of corneal allograft rejection


Autoři: Xiaoxiao Lu aff001;  Chenchen Chu aff001;  Xun Liu aff001;  Yichen Gao aff001;  Mianmian Wu aff001;  Fang Guo aff001;  Yahong Li aff001;  Chao Geng aff001;  Yue Huang aff001;  Yan Zhang aff001;  Shaozhen Zhao aff001
Působiště autorů: Tianjin Key Laboratory of Retinal Functions and Diseases, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, China aff001
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222515

Souhrn

Objective

The immune rejection mediated by CD4+ T cell and antigen presenting macrophages is the leading cause of corneal transplantation failure. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess robust immunomodulatory potentials, and have been shown by us and others to promote corneal allograft survival. However, the immunological mechanism underlying the protective effects of BM-MSCs remains unclear. Therefore, in the current study, this mechanism was investigated in a BM-MSC-treated rat model of corneal allograft rejection, in the hope to facilitate the search for novel interventional targets to corneal allograft rejection.

Methods

Lewis rats were subjected to corneal transplantation and then received subconjunctival injections of BM-MSCs (2×106 cells / 100 μl PBS) immediately and at day 3 post-transplantation. The control group received the injections of PBS with the same volume. The clinical parameters of the corneal allografts, including opacity, edema, and neovascularization, were regularly evaluated after transplantation. On day 10 post-transplantation, the corneal allografts were collected and subjected to flow cytometry and high-throughput RNA sequencing (RNA-seq). GO enrichment and KEGG pathways were analyzed. The quantitative realtime PCR (qPCR) and immunohistochemistry (IHC) were employed to validate the expression of the selected target genes at transcript and protein levels, respectively.

Results

BM-MSC subconjunctival administration prolonged the corneal allograft survival, with reduced opacity, alleviated edema, and diminished neovascularization. Flow cytometry showed reduced CD4+ T cells and CD68+ macrophages as well as boosted regulatory T cells (Tregs) in the BM-MSC-treated corneal allografts as compared with the PBS-treated counterparts. Moreover, the RNA-seq and qPCR results demonstrated that the transcript abundance of Cytotoxic T-Lymphocyte Associated Protein 4 (Ctla4), Protein Tyrosine Phosphatase, Receptor Type C (Ptprc), and C-X-C Motif Chemokine Ligand 9 (Cxcl9) genes were increased in the allografts of BM-MSC group compared with PBS group; whereas the expression of Heat Shock Protein Family A (Hsp70) Member 8 (Hspa8) gene was downregulated. The expression of these genes was confirmed by IHC at protein level.

Conclusion

Subconjunctival injections of BM-MSCs promoted corneal allograft survival, reduced CD4+ and CD68+ cell infiltration, and enriched Treg population in the allografts. The BM-MSC-induced upregulation of Ctla4, Ptprc, Cxcl9 genes and downregulation of Hspa8 gene might contribute to the protective effects of BM-MSCs and subserve the potential interventional targets to corneal allograft rejection.

Klíčová slova:

Cornea – Edema – Flow cytometry – Gene expression – T cells – Opacity – Corneal transplantation – Regulatory T cells


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