Impact of autophagy inhibition on targeting imaging of allotransplantation with ¹²⁵I-anti-TLR5

Objectives: It has been reported that TLR5 could radio-imaging allograft apparently, but whether Rapamycin (Rapa) activated autophagy affecting TLR5 targeting imaging is not clear. This study aimed to evaluate the effect of autophagy on TLR5-targeting imaging in allotransplantation mice model. Methods ¹²⁵I-anti-TLR5 was prepared and identified according to reference we published previously. Rapa was used to induce tolerance along with autophagy activating and 3MA was used as autophagy inhibitor. Allotransplantation mice model were established and randomly divided into four groups: Control, Rapa, 3MA and Rapa plus 3MA. The autophagy expression was measured by Western Blot. The uptake and dissociation of ¹²⁵I-anti-TLR5 with spleen cells of BALB/c mice was studied.¹²⁵I-anti-TLR5 was injected through tail veins on day 9 post allotranplantation. Biodistribution and whole-body phosphor-autoradiography were performed. T/NT ratio (Target to non-target, graft/opposite muscle) and radioactivity activity ratio in vivo were analyzed. Histology and immunohistochemical staining of Beclin-1 and TLR5 were performed on day 12 post transplantation. Results ¹²⁵I-anti-TLR5 was successfully prepared with 95.88% labeling rate and keep stable until 96h (89.04±1.16 % vs 87.44±0.58) in normal saline and serum. Autophagy molecules Beclin 1/ATG5 were increased after Rapa treated and decreased with 3MA administration. The uptake and dissociation of tracer was not apparently changed with the treatment of Rapa and/or 3MA compared with the Control. Biodistribution studies showed that ¹²⁵I-anti-TLR5 were mainly metabolized through the liver and kidney, and the high radioactivity in grafted skin was detected. The T/NT ratio was significantly higher in group of Rapa (13.25±0.14 vs 4.32±0.21, P<0.01) and Rapa plus 3MA (6.460±0.15 vs 4.32±0.21, P<0.01) than Control. Whole body phosphor-screen auto-radioimaging showed clear skin grafts from 48h, and radioactivity activity ratio remarkably increased with Rapa application in 72h (Rapa 5.33±0.12; and Rapa plus 3MA groups 2.46±0.38 vs Control 1.46±0.20, P<0.01, P<0.01). Acute inflammatory infiltration in allograft was apparently decreased in groups of Rapa and Rapa+3MA. Both TLR5 and Beclin-1 positive staining were increased in Rapa and Rapa+3MA groups compared with the group of Control, and no obviously difference was detected between the groups of Rapa+3MA and Rapa alone. Conclusion TLR5 expression is higher under Rapa application, autophagy induced by Rapa showed no significant impact on ¹²⁵I-anti-TLR5 targeted allograft imaging.

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