Aim: Antibody-mediated rejection (AMR) persists as a major issue affecting solid organ transplants. The purpose of this study was to investigate the proximal signaling events that regulate mTOR-mediated leukocyte recruitment following HLA I stimulation in endothelial cells (EC).

Methods: EC were treated with either pharmacological mTOR inhibitors, mTOR-pathway-directed siRNA, or Rho and ROCK inhibitors, then stimulated with HLA I antibodies. Phosphorylation of mTOR, ERK, ERM, and S6RP, as well as additional downstream effectors of the mTOR and Rho pathways were measured by Western blot. EC stimulated with thrombin served as positive control for mTOR-mediated signaling in WB experiments. Untreated and EC treated with an isotype control antibody served as negative controls.

Results: Using flow-based adherence assays to study the role of mTOR in monocyte recruitment, we previously discovered that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering, which has been attributed to Rho signaling. To examine the upstream/downstream relationship of RhoA and mTOR, we used the aforementioned inhibitors and measured mTOR phosphorylation, as well as the phosphorylation state of its downstream effector p70S6K. Notably, phosphorylation of mTOR following class I stimulation was not affected by RhoA inhibition. This data suggests that mTOR functions upstream of RhoA and that blocking RhoA through mTOR inhibition ablates its downstream effects, prevents focal adhesion assembly and ICAM-1 end stage clustering and firm adhesion. Given our findings that RhoA signals downstream of mTOR, we next examined the effects of mTOR inhibition on HLA-I induced ERM phosphorylation. We found that pharmacological and siRNA mTOR inhibition of HLA-I antibody-activated EC led to a significant reduction in ERM phosphorylation.

Conclusion: Our findings connect mTOR-mediated class I signaling to cytoskeletal rearrangements involving RhoA and ERM proteins. These observations suggest that mTOR inhibition reduces leukocyte recruitment to activated endothelium by impairing firm adhesion.

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