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

ABSTRACT: 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 ?l 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.

SUBMITTER: Lu X

PROVIDER: S-EPMC6756551 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Lu Xiaoxiao X Chu Chenchen C Liu Xun X Gao Yichen Y Wu Mianmian M Guo Fang F Li Yahong Y Geng Chao C Huang Yue Y Zhang Yan Y Zhao Shaozhen S

PloS one 20190923 9

<h4>Objective</h4>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- ...[more]

PMID: 31545822

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Project description:BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-? expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8?±?1.2 days). Injection twice of MSCs (MST 12.6?±?1.4 days) was more effective than a single injection (MST 10.8?±?1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4?+?T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-? expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Project description:Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro. Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68+ macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68+ macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft.

Project description:Background: Acute T cell-mediated rejection (TCMR) is a common immunological feature in renal transplantation and is associated with reduced graft survival. However, the full immunological profiling of acute TCMR remains unknown. Methods: A fully mismatched acute TCMR model was induced by transplanting the left kidneys from BALB/c mice into C56BL/6J mice. Allografted kidneys were harvested on day 7 (D7) and sorted for CD45+ leukocytes for single cell RNA sequencing (scRNA-seq) . In addition, scRNA-seq data of CD45+ immune cells on day 15 (D15) containing GSE157292 dataset (samples GSM4761000 and GSM4761003) were downloaded from Gene Expression Omnibus. All data obtained from D7 and D15 were integrated and analyzed. Results: We identified 20 major immune cell types among 10921 immune cells, including macrophages, B cells, T cells, dendritic cells, natural killer cells, neutrophils, and basophils in kidney allografts. Macrophages and CD8 T cells constituted the main immune cell populations on both D7 and D15. In the process towards immune regression, the proportion of CD8 T cells dropped significantly by about 25%, mainly due to decreased proliferation and naïve CD8 T cell infiltration. The proportion of both B cells and neutrophils also decreased by about 3 folds. On the contrary, the relative proportion of macrophages and DCs increased significantly, especially by a 4 fold increase in Ly6cloMrc1+ resident macrophages A group of Ly6cloEar2+ macrophages also increased by about 3 folds. Moreover, myeloid cells harbored the richest ligand and receptor (LR) pairs with other cell types, particularly for chemokine ligands such as Cxcl9, Cxcl10, Cxcl16 and Yars. However, macrophages with weak response to interferon gamma (IFNg) contributed to rejection regression. Conclusions: There is a comprehensive single-cell landscape of the kidney allograft immune cells from rejection progression to regression in acute TCMR. Reduction in CD8 T cells, B cells, and neutrophils while increasing in Ly6clo macrophages, including Ly6cloMrc1+ resident macrophages and Ly6cloEar2+ macrophages, may contribute significantly to immune rejection regression during acute TCMR.

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

Publications

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

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