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Abstract
ObjectiveThe 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.MethodsLewis 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.ResultsBM-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.ConclusionSubconjunctival 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.
Highlights
Corneal transplantation is the main therapeutic modality to corneal blindness[1]
The BM-MSCinduced upregulation of Cytotoxic T-Lymphocyte Associated Protein 4 (Ctla4), Ptprc, Cxcl9 genes and downregulation of Hspa8 gene might contribute to the protective effects of Bone marrow-derived mesenchymal stem cells (BM-mesenchymal stem cells (MSCs)) and subserve the potential interventional targets to corneal allograft rejection
In the phosphate buffered solution (PBS) group, there were more neo-vessels growing into the corneal grafts and reaching the graft center than the BM-MSC group at day 10 post-operation
Summary
Corneal transplantation is the main therapeutic modality to corneal blindness[1]. To certain extent, are protected by ocular immune privileges, immune rejection remains the leading cause of keratoplasty failure[2]. Accumulating evidences indicate that the immune responses mediated by CD4+ T cells and antigen presenting macrophages play critical roles in launching the immune rejection[3]. The immunosuppressive agents, including Cyclosporin A, FK506, and glucocorticoids, can inhibit immune responses, subdue inflammatory cell infiltration, and prolong corneal allograft survival[4, 5]. Long-term use of these immunosuppressives is associated with severe side effects, such as cataract and elevated intraocular pressure, which profoundly limit their clinical applications[6]. A new, effective, and safe therapeutic modality is needed to inhibit immune rejection and promote corneal allograft survival
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