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. 2021 Oct 19:12:778892.
doi: 10.3389/fphar.2021.778892. eCollection 2021.

Treatment With Melatonin After Corneal Graft Attenuates Rejection

Ziqian Zhu  1 , Ruiping Peng  1 , Hongyi Shen  1 , Lei Zhong  1 , Siqi Song  1 , Tao Wang  1 , Shiqi Ling  1
Affiliations

Treatment With Melatonin After Corneal Graft Attenuates Rejection

Ziqian Zhu et al. Front Pharmacol. .

Abstract

Background: Immunologic graft rejection is the main complication of corneal transplants. This study aimed to investigate the effect of melatonin (MT) on the rejection of corneal transplantation. Methods: Corneal allografts were performed by grafting corneas from BALB/C mice to C57BL/6 hosts. MT (50 mg/kg) was intraperitoneally injected into the hosts every day from the day of transplantation. The survival of grafts was observed by slit lamp biomicroscopy, and inflammatory cell infiltration was detected by hematoxylin and eosin staining and immunohistochemistry. The balance of Teff and Treg immune cells in draining lymph nodes (DLNs) was detected by flow cytometry. The levels of cytokines related to the grafts and DLNs were detected using real-time fluorescence quantitative PCR. Additionally, we used the mouse macrophage line RAW264.7 to study the effect of MT on the activation of NLRP3 inflammatory body. Results: MT treatment improved the graft survival rate, reduced inflammatory cell infiltration in the graft, decreased the percentage of Th1/Th17 cells in the DLNs, and increased the percentage of Treg cells. Melatonin inhibited the activation of the NLRP3 inflammasome, thereby reducing the expression of IL-1β and other related proinflammatory cytokines such as MCP-1, MIP-1, NLRP3, ASC, TNF-a and VEGF-A (all p < 0.05). Conclusion: Our study demonstrates that MT promotes the survival of mouse corneal grafts by inhibiting NLRP3-mediated immune regulation, reducing immune cell activation and cell migration, and inhibiting the production of inflammatory-related cytokines. Treatment with MT might provide a potential clinical therapeutic target for corneal transplantation.

Keywords: CD4+ T cells; NLRP3 inflammasome; corneal transplant; macrophages; melatonin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with the authors at the time of the review.

Figures

FIGURE 1
FIGURE 1
MT therapy alleviates corneal graft rejection and corneal lymphangiogenesis and angiogenesis. (A) Establishment of the corneal transplantation model. (B) Combined image of all lymphatic vessels reaching the interface 9 days after corneal transplantation (white arrows). (C) Postoperative corneal graft survival curve of mice (CT; MT:50 mg/kg). MT injection delayed corneal graft rejection compared to the saline group (n = 8 per group). (D) On day 9 of transplantation, LYVE-1 and CD31+ staining (white arrows) were performed on the entire cornea of mice. In the normal mouse eye, lymphatics and neovessles are located only at the margin of the cornea and the corneal lymphangiogenesis and angiogenesis developed after transplantation (Scale: 100 µm). (E) MT inhibited corneal lymphangiogenesis and angiogenesis (n = 3 per group) (Nor: Normal group; Con: control group; MT: treated with MT). *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 2
FIGURE 2
MT inhibits inflammatory cells, F4/80, and CD11b recruitment. (A) At day 9 of transplantation, histopathology revealed less inflammatory cell (white arrows) infiltration in corneal grafts in the MT group (representative images for each group). Fluorescence images showing infiltration of F4/80 and CD11b in the grafts of each group at day 9. Corneal sections were stained with immunofluorescent antibodies for positive cells, as shown (Scale: 50 µm). (B) Count of positive cells in corneal grafts of each group on day 9 (n = 3 per group). (Nor: Normal group; Con: control group; MT: treated with MT). *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3
FIGURE 3
MT inhibits the expression of chemokines and proinflammatory factors in the cornea. On the 9th day after surgery, the mRNA expression levels of relevant cytokines in the grafts were determined by quantitative RT-PCR (n = 4 per group). (Nor: Normal group; Con: control group; MT: treated with MT). *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 4
FIGURE 4
MT regulates Teff/Treg balance in corneal graft model mice. (A–F) On the 9th day after operation, the levels of Teff/Treg cells in DLNs were measured by flow cytometry. MT was found to inhibit the proportion of Th1/Th17 cells in DLNs (n = 6 per group). (G–H), while treatment increased the proportion of Treg cells in DLNs (n = 6 per group). (I–L) Real-time quantitative PCR was used to measure the expression level of inflammatory related mRNA. MT inhibited the mRNA expression of IL-17, IFN-γ, TNF-α, and Foxp3 (n = 4 per group). (Con: control group; MT: treated with MT). *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 5
FIGURE 5
MT inhibits the expression of inflammatory cytokines in RAW264.7 macrophages. (A) mRNA expression of inflammatory cytokines was measured by real-time quantitative PCR (n = 4 per group). (B) The cell viability was determined by CCK8 assay (n = 4 per group). (C) Protein expression of inflammatory cytokines in culture supernatants was measured by ELISA (n = 4 per group). (Nor: Normal group; Con: control group; MT: treated with MT). *p < 0.05, **p < 0.01, ***p < 0.001.
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