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  • Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

Investigative ophthalmology & visual science (2021-06-02)
Jun Zhu, Takenori Inomata, Keiichi Fujimoto, Koichiro Uchida, Kenta Fujio, Ken Nagino, Maria Miura, Naoko Negishi, Yuichi Okumura, Yasutsugu Akasaki, Kunihiko Hirosawa, Mizu Kuwahara, Atsuko Eguchi, Hurramhon Shokirova, Ai Yanagawa, Akie Midorikawa-Inomata, Akira Murakami
ABSTRACT

To investigate the effects of ex vivo-induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation. Bone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription-quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo-induced BM-MDSCs. IFN-γ, IL-2, IL-10, and TGF-β1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo-induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry. The combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo-induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo-induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-β1 production was observed in coculture of ex vivo-induced BM-MDSCs. Injected ex vivo-induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis. The ex vivo-induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
NG-Monomethyl-L-arginine, Monoacetate Salt, Cell permeable. L-Arginine analog that acts as a competitive inhibitor of all three isoforms of NOS.
Sigma-Aldrich
Nω-Hydroxy-nor-L-arginine, Diacetate Salt, A potent, selective, competitive, and high affinity inhibitor of arginase.