AUTOPHAGY EXPLORES THE MECHANISM OF IL-1Β INVOLVED IN CORNEAL ALLOGRAFT REJECTION BY REGULATING NLRP3 INFLAMMATORY BODIES

Lin Li^{1, #, *}, Gang An^{2, #}, Xiaoguang Zhang³, Lu Lu²

¹Department of Ophthalmology, Zhejiang Jinhua Eye Hospital, Jinhua, PR China - ²Department of Ophthalmology, Liaoning Jinzhou Central Hospital, Jinzhou, PR China - ³Department of Ophthalmology, General Hospital of Northern Theater Command of China PLA (Heping Compang), Shenyang, PR China

Objective: To investigate the role of autophagy in the immune rejection of corneal transplantation by regulating the nucleotide binding oligomerization domain-like NLR family pyrin domain containing 3 (NLRP3) inflammatory bodies mediated by interleukin-1β (IL-1β) involved in corneal allograft rejection.

Methods: Thirty clean and healthy male BALB/c mice and C57BL/6 mice were selected. The corneal transplantation mice were randomly divided into three groups: an allogeneic transplantation group, a rapamycin (rapa) eye drop group and a 3-methyladenine (3-mA) eye drop group. BALB/c mice were the recipients in the allogeneic transplantation group, Rapa eye drop group and 3-mA eye drop group, and C57BL 6 mice were the donors. The corneal transplantation model was established in an NLRP3 gene deficient group and wild-type mice. The recipient of the NLRP3 gene deficient group was NLRP3 gene deficient mice, and the donor was C57BL/6 mice. The donor corneal grafts were transplanted on the recipient bed. The eyes were smeared with ofloxacin eye cream after the keratoplasty. The Rapa eye drop group and 3-mA eye drop group began to take the eyes after removing the eyelid suture one day after the operation, three times a day. Fifteen rats in each group were selected the next day to observe the oedema and turbidity of corneal grafts and the growth of neovascularization in each group (allogeneic transplantation group, Rapa eye drop group, 3-mA eye drop group, NLRP3 gene defect group, wild type group). The HE staining method was used to observe changes in corneal pathology in mice in each group one month after operation. The expression of p62, LC3-I and LC3-II in mice of the allogeneic transplantation group, Rapa eye drop group and 3-mA eye drop group were observed with Western blotting. The levels of IL-1 β in serum from the allogeneic transplantation group, Rapa eye drop group and 3-mA eye drop group were measured by enzyme-linked immunosorbent assay, and the expression of IL-1 β in the lysate of mice in each group (allogeneic transplantation group, Rapa eye drop group, 3-mA eye drop group, NLRP3 gene defect group, wild type group) was measured with Western blotting. 

Results: Compared with the allogeneic transplantation group, there was no immune transplantation reaction in the Rapa group: the corneal graft was transparent, without oedema and turbidity, and there was no obvious neovascularization in the corneal graft area. In the 3-mA eye drop group, there was an immune transplantation reaction, with corneal graft oedema and thickening, turbidity, and obvious neovascularization in the corneal graft area. Compared with the allogeneic transplantation group, there was no obvious oedema in the Rapa eye drop group and less inflammatory cell infiltration in the corneal stroma; the 3-mA eye drop group had obvious oedema and a large number of inflammatory cell infiltrations in the corneal stroma. Compared with the wild-type group, NLRP3 gene deficient mice had no obvious oedema and fewer inflammatory cell infiltrations in the corneal stroma. Compared with the allogeneic transplantation group, the expression level of p62 in the Rapa eye drop group was significantly lower, and the expression level of LC3-II was significantly higher than the LC3-I. The expression level of p62 in the 3-mA eye drop group was significantly higher, and the expression level of LC3-II was significantly lower than that of LC3-I. Compared with the allogeneic transplantation group, the level of IL-1 β in the serum and lysate of the mice in the Rapa eye drop group was significantly lower, while the level of IL-1 β in the 3-mA eye drop group was significantly higher. The expression level of IL-1 β in the lysate of NLRP3 gene deficient mice was significantly lower than that of wild type mice. 

Conclusion: NLRP3 inflammatory bodies participate in corneal transplantation rejection and inhibit the transformation of IL-1 β from precursor to mature. Autophagy can mediate the involvement of IL-1 β in the inhibition of corneal allograft rejection through the regulation of NLRP3 inflammatory bodies.