Protein phosphatase 2A activation promotes heart transplant acceptance in mice

Background: While heart transplantation is the standard treatment for heart failure, both acute and chronic transplant rejection frequently occur. Since the modulation of protein phosphatase PP2A activity is critical for tissue and organ homeostasis under physiological and pathophysiological conditions, PP2A may also affect the ability to tolerate transplanted organs. Here we demonstrate that administration of a novel class of small molecule activators of PP2A (SMAPs) prolonged cardiac allograft survival in a mouse heterotopic heart transplantation model. Mechanistically, SMAPs effectively suppressed the inflammatory immune response while increasing Treg population in the allografts, findings corroborated by functional analysis of RNAseq data derived from Tregs of treated splenic tissue. Importantly, SMAPs further prolonged CTLA4-Ig (an immunosuppressive agent utilized in organ transplantation)-induced cardiac rejection tolerance and extended allograft survival. SMAPs also strongly mitigated cardiac allograft vasculopathy as evidenced by a marked reduction of neointimal hyperplasia and smooth muscle cell proliferation. Mechanistic studies implicate SMAPs elicited suppression of MEK/ERK pathways as a unifying mechanism for the aforementioned effects in Tregs and SMCs. These findings highlight the potential of PP2A activation in improving alloengraftment in heart transplantation and add knowledge to the phosphatase driven regulation of the immune system in the context of organ transplantation. Male C57BL/6 (B6, H-2b) and BALB/c (B/c, H-2d) mice aged 12–16 weeks were used in this study. Donor hearts from BALB/c male mice were transplanted onto the inferior vena cava and aorta in the peritoneal cavity of male C57BL/6J mice. Syngeneic heart graft is defined as a donor heart from C57BL/6J male mice transplanted into C57BL/6J male mice. A 5 mg/kg dose of DT-061 was given intragastrically twice a day beginning on the day of transplantation and continued until the completion of experiments. After 7 days, CD3+CD4+CD25+CD127low cells (Tregs) were sorted by flow cytometry from the spleens of vehicle group and the DT-061 treated group and sent for RNA-seq study.

研究背景：尽管心脏移植是心力衰竭的标准治疗手段，但急性与慢性移植排斥反应仍频繁发生。鉴于蛋白磷酸酶PP2A（protein phosphatase PP2A）的活性调控在生理及病理生理条件下对组织与器官稳态至关重要，其或可影响移植器官的耐受能力。本研究证实，新型PP2A小分子激活剂（SMAPs）可延长小鼠异位心脏移植模型的心脏同种异体移植物存活时间。机制层面，SMAPs可有效抑制炎症性免疫应答，同时增加移植物内调节性T细胞（Treg）群体，该结论通过对给药组脾脏来源Treg的RNA测序（RNA-seq）数据进行功能分析得到验证。值得注意的是，SMAPs可进一步延长细胞毒性T淋巴细胞相关抗原4-免疫球蛋白（CTLA4-Ig，一种器官移植中使用的免疫抑制剂）诱导的心脏移植排斥耐受，并延长同种异体移植物存活时长。此外，SMAPs可显著缓解心脏同种异体移植血管病，表现为内膜增生与平滑肌细胞增殖均显著减少。机制研究显示，SMAPs介导的丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶（MEK/ERK）通路抑制，是其在调节性T细胞与平滑肌细胞中发挥上述作用的共同机制。本研究结果凸显了PP2A激活在改善心脏移植同种异体植入方面的应用潜力，并丰富了器官移植场景下磷酸酶介导的免疫系统调控相关认知。
本研究使用12~16周龄的雄性C57BL/6（B6，H-2b）与BALB/c（B/c，H-2d）小鼠。将BALB/c雄性小鼠的供体心脏移植至雄性C57BL/6J小鼠腹腔内的下腔静脉与主动脉处。同基因心脏移植物定义为：供体心脏来自C57BL/6J雄性小鼠，并移植至C57BL/6J雄性小鼠体内。于移植当日开始，以5 mg/kg剂量的DT-061每日两次灌胃给药，持续至实验结束。给药7天后，通过流式细胞术从溶剂对照组与DT-061处理组小鼠的脾脏中分选CD3+CD4+CD25+CD127low细胞（调节性T细胞，Treg），并送检进行RNA-seq测序分析。