Mechanism of immune tolerance induced by donor derived immature dendritic cells in rat high-risk corneal transplantation

Abstract

Aim: To study the role of immature dendritic cells (imDCs) on immune tolerance in rat penetrating keratoplasty (PKP) in high-risk eyes and to investigate the mechanism of immune hyporesponsiveness induced by donor-derived imDCs.

Methods: Seventy-five SD rats (recipient) and 39 Wistar rats (donor) were randomly divided into 3 groups: control, imDC and mature dendritic cell (mDC) group respectively. Using a model of orthotopic corneal transplantation in which allografts were placed in neovascularized high-risk eyes of recipient rat. Corneal neovascularization was induced by alkaline burn in the central cornea of recipient rat. Recipients in imDC group or mDC group were injected donor bone marrow-derived imDCs or mDCs of 1×10(6) respectively 1 week before corneal transplantation via tail vein. Control rat received the same volume of PBS. In each group, 16 recipients were kept for determination of survival time and other 9 recipients were executed on day 3, 7 and 14 after transplantation. Cornea was harvested for hematoxylin-eosin staining and acute rejection evaluation, Western blot was used to detect the expression level of Foxp3.

Results: The mean survival time of imDC group was significantly longer than that of control and mDC groups (all P<0.05). The expression level of Foxp3 on CD4(+)CD25(+)T cells of imDC group (2.24±0.18) was significantly higher than that in the control (1.68±0.09) and mDC groups (1.46±0.13) (all P<0.05).

Conclusion: Donor-derived imDC is an effective treatment in inducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance induced by imDC might be inhibit T lymphocytes responsiveness by regulatory T cells.

Keywords: high-risk keratoplasty; immature dendritic cell; regulatory T cells.

Figure 1. Morphological appearance of imDC and mDC Phase-contrast microscopy showed visible DCs morphological rules

A: The main cell is small round or oval in imDCs, cells formed small clusters composed of four to six; B: The mDCs have long, slender dendrites. These dendrites can extend several times the length of the cell body, and are reminiscent of the typical morphology; C: Scanning electron microscopy showed the branches sample protuberance short and less in imDC; D: mDC form prominent dendrites and have long cytoplasmic veils by scanning electron microscopy.

Figure 2. Expression of cell surface molecules on DCs

After 6 days in culture in GM-CSF and IL-4–supplemented medium, the loosely attached clusters were collected as imDCs. mDCs were generated from BM cells cultured with rrGM-CSF for 6 days followed by stimulation with TNF-α for 48 hours. DCs were pooled for flow cytometry.

Figure 3. The graft survival in different groups

After surgery, heavy corneal edema was seen in control group at the time of postoperative day 14 (A). enlarged blood vessels were seen around the graft in mDC group (C). In imDC group, the graft remained clear, with less neovasculature moving into the grafts(middle).

Figure 4. Survival curves for different groups

Three experimental groups were distinguished: comparison of allograft survival between the three of the control group (MST, 9.0±0.46 days, n=16), imDC group (MST, 18.25±0.68 days, n=16) and mDC group (MST, 7.63±0.49 days, n=16).

Figure 5. The hematoxylin-eosin staining of allografts from different groups

The allografts of the control (A) and mDC (C) group rats at the time of postoperative day 14 had edema and infiltration of cells, especially in the stroma, and there were lot of new blood vessels. The corneal neovascularization was reduced in imDC group (B) and do not have much mononuclear cell infiltration.

Figure 6. Foxp3 proteins detection by Western blot assay

Line 1 to 3 was the expression of Foxp3 on postoperative day 3 (control group; imDC group; mDC group); Line 4 to 6 was the expression of Foxp3 on postoperative day 7 (control group; imDC group; mDC group); Line 7 to 9 was the expression of Foxp3 on postoperative day 14 (control group; imDC group; mDC group), Foxp3 can be detected in every groups and these higher molecular weight bands are only present in the imDC groups.