白芍总苷基于JAK/STAT6通路调控巨噬细胞PD-L2表达促进狼疮性肾炎免疫耐受的机制研究

Macrophages play a key role in regulating immune response and the inducing of macrophage M2 polarization represents a potential therapeutic strategy for inducing Lupus nephritis (LN) immunotolerance. PD-L2 is one of the main immune regulatory for the maintenance of peripheral tolerance. Our previous studies t have demonstrated that PD-L2 is specifically expressed in macrophages and the macrophage expression of PD-L2 in LN mice is much lower compared with that in control mice. Total glucosides of paeony (TGP) could effectively ameliorate the renal pathologic injury in LN mice, accompanying with a sharp increase in PD-L2+ M2 macrophage. Moreover, we found that the effect of TGP on macrophage PD-L2 expression is associated with the regulation of JAK/STAT6 pathway in vitro. Based on these findings, we hypothesized the expression of PD-L2 is beneficial to. And TGP induces the PD-L2 expression in macrophage through the JAK/STAT6 pathway, thus promoting the effect of macrophage on inducing LN immunotolerance. Therefore, multiple techniques including in-vitro cell co-culture model, macrophage-based immunotherapy, spontaneous LN mode and in vitro and in vivo gene modification will be utilized to clear the pivotal role of PD-L2 in regulating M2 macrophage functions basing on the effect of phagocytosis and maintaining T lymphocyte balance in macrophage. And then to explore the therapeutic mechanism of TGP on LN based on the JAK/STAT6/PD-L2 pathway in macrophage. The study will provide better understanding of molecular mechanisms underlying LN treatments with TGP, and help develop new strategies for the treatment and prevention of LN.

巨噬细胞是调控免疫应答的中心环节，诱导巨噬细胞M2极化可促进狼疮性肾炎（LN）免疫耐受，改善LN病理损伤。PD-L2是免疫耐受的重要诱导分子，我们前期研究发现，PD-L2特异性表达于M2巨噬细胞，LN小鼠巨噬细胞PD-L2表达较对照小鼠明显降低，而白芍总苷可上调巨噬细胞PD-L2表达，促进LN免疫耐受。进一步研究发现，白芍总苷上调PD-L2表达与JAK/STAT6通路相关。由此，我们提出“白芍总苷可通过JAK/STAT6通路诱导巨噬细胞PD-L2表达，进而促进巨噬细胞诱导LN免疫耐受”。本课题拟应用巨噬细胞/T细胞共培养、巨噬细胞免疫治疗、LN自发模型及体内外基因重组等手段，从巨噬细胞吞噬凋亡细胞及调节T细胞免疫平衡探讨PD-L2对巨噬细胞功能的调节机制，进而从巨噬细胞JAK/STAT6/PD-L2通路揭示白芍总苷诱导LN免疫耐受的作用机制。以期为LN病机拓展新视野，为LN防治提供新策略。

大量证据表明巨噬细胞M2极化对慢性肾脏疾病患者有益，已有报道PD-1配体(PD-Ls)参与巨噬细胞M2极化及其免疫抑制作用。白芍总苷(TGP)目前已被批准用于治疗某些自身免疫性疾病。我们在狼疮性肾炎小鼠模型上研究了TGP对狼疮性肾炎的潜在治疗作用，并探讨了其诱导Treg分子机制。我们发现PD-L2更多表达在M2巨噬细胞中，白芍总苷可通过激活STAT6通路上调巨噬细胞PD-L2表达。PD-L2+ M2巨噬细胞比PD-L2- M2巨噬细胞具有更强的诱导Treg分化及增殖的作用。PD-L2+ M2巨噬细胞治疗可以改善狼疮性肾炎小鼠肾功能，并能在体诱导Treg。TGP治疗能显著改善肾功能，降低尿蛋白和血肌酐，降低血清抗ds-DNA水平，减少肾脏补体沉积，改善肾脏病理；白芍总苷治疗可抑制LN小鼠效应T细胞，诱导Treg细胞；另外，TGP可增加脾脏和腹腔及肾脏巨噬细胞M2极化。重要的是，TGP治疗提高了脾脏、腹腔灌洗液和肾脏中PD-L1/2+巨噬细胞群比；STAT6抑制剂联合应用可在体阻断TGP介导的Treg分化及抑制效应T细胞的作用，同时STAT6抑制剂可抑制TGP介导LN小鼠巨噬细胞PD-L2表达上调，而对巨噬细胞PD-L1表达无明显作用。基于此，我们从巨噬细胞STAT6/PD-L2通路探讨了白芍总苷治疗LN的作用机制，并阐释了PD-L2在巨噬细胞诱导Treg细胞中的关键作用。