TLR3-overexpressing umbilical cord mesenchymal stromal cells suppress immune responses to attenuate high-risk corneal transplantation rejection

Abstract

Background: The success rates of high-risk corneal transplantation are significantly hindered by immunological rejection. Umbilical cord mesenchymal stem cells (UC-MSCs) have emerged as a potential solution to improve graft outcomes. Toll-like receptors (TLRs) play pivotal roles in the immune response, however, their specific functions in UC-MSC-based treatments for corneal graft rejection requires further investigation.

Methods: New Zealand rabbits that underwent high-risk corneal transplantation were treated with UC-MSC-coated contact lenses (MSCohi-O), while blank lenses and untreated grafts served as controls. Clinical manifestations related to rejection were assessed. RNA-seq was performed on the grafts. GSEA, CIBERSORT, and flow cytometry analyses were performed to investigate the activation of signaling pathways and immune changes. The microarray dataset GSE68610, which contains transcriptome data for normal human UC-MSCs and UC-MSCs treated with cytokines, was analyzed to evaluate TLR expression and identify the key factors involved in the anti-inflammatory effect of UC-MSCs. Overexpression and knockout of TLR3 were performed in UC-MSCs, and the therapeutic effects of TLR3-activated and -inactivated UC-MSCs were compared in a high-risk corneal transplantation model.

Results: MSCohi-O treatment alleviated corneal opacity and edema, inhibited neovascularization, promoted epithelialization, and prolonged the survival time of corneal grafts (all p < 0.05). GSEA revealed that the allograft rejection pathway was upregulated in untreated grafts and downregulated in UC-MSC-treated grafts. Increased numbers of Tregs and decreased numbers of Th17 cells were observed in UC-MSC-treated corneas. An analysis of the GSE68610 dataset revealed that TLR3 expression was upregulated in cytokine-activated UC-MSCs, suggesting that TLR3 is a potential regulator of the immunosuppression function of UC-MSCs. TLR3-overexpressing UC-MSCs exhibited enhanced suppression of rejection in high-risk corneal transplants, whereas TLR3 knockdown diminished these effects.

Conclusions: This study shows that the localized application of UC-MSC-coated contact lenses is capable of inhibiting rejection in high-risk corneal transplantation. TLR3-overexpressing UC-MSCs have enhanced antirejection and immunomodulatory effects. This research could offer a safer and more effective therapeutic strategy to prevent corneal transplant rejection.

Keywords: Corneal transplantation rejection; Immune; Mesenchymal stromal cells; TLR3.

Fig. 1

UC-MSCs alleviated corneal edema, inflammation, and corneal epithelial defects after high-risk corneal transplantation. (A) Schematic representation of the high-risk rejection model construction, treatment, and examinations. (B) Representative image of a normal cornea from a New Zealand rabbit. (C) Representative image of neovascularization induced by sutures. (D) Diffused light, slit light, and corneal fluorescein staining under a slit lamp. (E) Representative image of AS-OCT of the different groups and the corneal thickness evaluation. (F) Corneal opacity scores of the different groups. (G) Proportions of corneal epithelial defects in the different groups. (H) Inflammation indices of the different groups. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 2

UC-MSCs inhibited high-risk corneal graft rejection. (A) Representative images of H&E staining. (B) Rejection indices (left panel) and survival curves (right panel) of the different groups. (C) Schematic representation of the RNA-seq data. (D) Volcano plot of DEGs between the untreated and normal groups and between the MSCohi-O and blank lens groups. (E) Ridgeline plot showing the GSEA of DEGs between the untreated and normal groups. (F) The allograft rejection pathway, graft-versus-host disease pathway, and antigen processing and presentation pathway were upregulated in untreated grafts. (G) Ridgeline plot showing the GSEA of DEGs between the MSCohi-O and blank lens groups. (H) The allograft rejection pathway, graft-versus-host disease pathway, cytokine–receptor interaction pathway, and cytokine activity pathway were downregulated. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 3

UC-MSCs attenuated the immunological process of rejection. (A) Bar plot of 22 types of immune cells in normal and rejected corneas. (B) Bar plot of 22 types of immune cells in blank lens-treated and MSCohi-O treated corneas. (C) Immune cells undergoing changes within rejected corneas. (D) Changes in immune cells undergoing changes within MSCohi-O-treated corneas. (E) GSEA revealed upregulated Th cell-related pathways in rejected grafts. (F) GSEA revealed downregulated Th cell-related pathways in UC-MSC-treated grafts. (G) Flow cytometry detection of Th17 cells and Tregs in different groups. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

TLR3 was identified as a potential regulator of the immunosuppressive function of UC-MSCs. (A) Heatmap of the DEGs between cytokine-stimulated UC-MSCs and normal UC-MSCs. (B) Schematic representation of the different roles of TLRs in modulating UC-MSC functions. (C) Heatmap of the differentially expressed TLRs between cytokine-stimulated UC-MSCs and normal UC-MSCs. (D) TLR2 and TLR3 were upregulated in activated UC-MSCs. (E) Western blots of different UC-MSCs. The image shown is a cropped membrane that depicts the proteins of interest. The full-length blots are presented in the Additional file. (F) Representative images of the immunofluorescence staining of different UC-MSCs. (G) Characterization of different UC-MSCs by flow cytometry through labeling MSC markers. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 5

TLR3 is a key molecule involved in the UC-MSC-mediated treatment of high-risk corneal transplant rejection. (A) Schematic representation of the treatment with TLR3-edited UC-MSCs. (B) Representative image of AS-OCT of the different groups and the corneal thickness evaluation. (C) Representative images of diffuse light and slit light of grafts under a slit lamp. (D) Representative images of corneal fluorescein staining under a slit lamp. (E) Representative images of H&E staining. (F) Corneal opacity scores of the different groups. (G) Proportion of corneal epithelial defects in the different groups. (H) Inflammation indices of the different groups. (I) Rejection indices of the different groups. (J) Survival curves of the different groups. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

IL-17 signaling is a potential mechanism underlying the TLR3-dependent antirejection effects of UC-MSCs. (A) GSEA revealed the inhibition of the IL-17 signaling pathway in the MSCohi-O group. (B) Schematic representation of IL-17 signaling and immunomodulation. (C) Representative images of immunohistochemical staining for IL-17 in the normal, untreated, blank lens, and MSCohi-O groups. (D) Representative images of immunohistochemical staining for IL-17 in the blank lens, TLR3-OE MSC, OE-ctrl MSC, sg-ctrl MSC, and sg-TLR3 MSC groups. (E) RT‒PCR was performed to determine the relative expression level of IL-17 in the different groups. (F) Flow cytometry detection of Th17 cells in different groups. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 7

TLR3 enhanced PGE2 secretion and mediated inhibition of STAT3 effects of UC-MSCs. (A) ELISA was performed to determine the secretion level of PGE2 in the different UC-MSCs groups. (B) RT‒PCR was performed to determine the relative expression level of PTGES in the different UC-MSCs groups. (C) RT‒PCR was performed to determine the relative expression level of STAT3 in the different corneal grafts (D) Representative images of immunohistochemical staining for STAT3 in the blank lens, TLR3-OE MSC, OE-ctrl MSC, sg-ctrl MSC, and sg-TLR3 MSC groups. The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001