Innate Immune Response Analysis in Meniscus Xenotransplantation Using Normal and Triple Knockout Jeju Native Pigs

Abstract

Although allogenic meniscus grafting can be immunologically safe, it causes immune rejection due to an imbalanced tissue supply between donor and recipient. Pigs are anatomically and physiologically similar to adult humans and are, therefore, considered to be advantageous xenotransplantation models. However, immune rejection caused by genetic difference damages the donor tissue and can sometimes cause sudden death. Immune rejection is caused by genes; porcine GGTA1, CMAH, and B4GLANT2 are the most common. In this study, we evaluated immune cells infiltrating the pig meniscus transplanted subcutaneously into BALB/c mice bred for three weeks. We compared the biocompatibility of normal Jeju native black pig (JNP) meniscus with that of triple knockout (TKO) JNP meniscus (α-gal epitope, N-glycolylneuraminic acid (Neu5Gc), and Sd (a) epitope knockout using CRISPR-Cas 9). Mast cells, eosinophils, neutrophils, and macrophages were found to have infiltrated the transplant boundary in the sham (without transplantation), normal (normal JNP), and test (TKO JNP) samples after immunohistochemical analysis. When compared to normal and sham groups, TKO was lower. Cytokine levels did not differ significantly between normal and test groups. Because chronic rejection can occur after meniscus transplantation associated with immune cell infiltration, we propose studies with multiple genetic editing to prevent immune rejection.

Keywords: immune rejection; meniscus; triple knockout; xenotransplantation.

Figure 1

Procedures for meniscus transplantation of normal/TKO JNP and graft survival rate. (A) The meniscus from the normal and TKO JNP groups was removed and divided into sections measuring about 50 mm³. (B) Mice were given isoflurane anesthesia while the meniscus was implanted in the dorsal back dermis. (C) After three weeks of breeding, the mice were euthanized for additional histological analysis. (D) Meniscus graft survival rate was illustrated.

Figure 2

Histological analysis of infiltrated cells in the sham, normal, and TKO JNP groups. The meniscus is extracted from the normal and TKO JNP groups and was cut into sections of approximately 50 mm³. The meniscus was transplanted in the dorsal back dermis of mice. The mice were bred for about three weeks. The mouse dermis containing the pig meniscus and sham was examined by H&E (A) and Safranin O staining (B). E: epidermis; D: dermis; M: meniscus. Magnification (upper panel: 100X, lower panel: 400X). Scale bars: 50 μm.

Figure 3

Immunohistochemical analysis of mast cell activation in the sham, normal and TKO JNP groups. The distribution of mast cells was partially confirmed by Alcian blue staining (A), and the quantification of mast cells in each group is represented (B). In each field, mast cell count for the sham (n, observed field = 7), normal JNP (n, observed field = 18), and TKO JNP (n, observed field = 13) were determined. A two-tailed independent t-test was used for the statistical analysis. E: epidermis; D: dermis; M: meniscus. Magnification (upper panel: 100X, lower panel: 400X). Scale bars: 50 μm.

Figure 4

Immunohistochemical analysis of eosinophil and extracellular trap (NET) infiltration in the sham, normal, and TKO JNP groups. The distribution of eosinophils and NET was analyzed by the Congo red (A) and CDr15 (C) staining. The quantification of eosinophils (B) and NET (D) is represented. In each field, eosinophil count for the sham (n = 10), normal (n = 21), and TKO JNP (n = 20) groups; and NET count for the sham (n = 6), normal (n = 21), and TKO JNP (n = 14) groups were determined. A two-tailed independent t-test was used for statistical analysis. E: epidermis; D: dermis; M: meniscus. Magnification of A (upper panel: 100X, lower panel: 400X) and C (200X). Scale bars: 50 μm. DAPI (blue for nucleus), CDr15 (Yellow for neutrophil extracellular trap), Merge (DAPI + CDr15).

Figure 5

Immunohistochemical analysis of macrophage infiltration in the sham, normal, and TKO JNP groups. The distribution of macrophages was analyzed by anti-F4/80 antibody staining (A), and the fluorescence intensity of F4/80 was represented in (B). In each field, macrophage counts for the sham (n = 4), normal (n = 11), and TKO JNP group (n = 10) were determined. A two-tailed independent t-test was used for significant analysis. Magnification: 200X. Scale bars: 50 μm. DAPI (blue for nucleus), F4/80 (Yellow for macrophage), Merge (DAPI + F4/80).