Blocking TLR-Activation of Regulatory T cells Slows Disease in ALS

描述(由申请人提供)：阻断调节性T细胞的TLR激活可减缓肌萎缩侧索硬化症的疾病。肌萎缩侧索硬化症(ALS)是一种毁灭性的、进展迅速的神经退行性疾病。我们最近的数据表明，调节性T细胞(Tregs)在ALS患者和ALS突变(M)SOD1小鼠模型中都减缓了疾病进展速度。在ALS患者中，进展较快的患者外周血中Tregs的数量较进展缓慢的患者减少，CD25、FoxP3、IL4、TGFβ的表达和CD25、FoxP3、IL4的mRNA表达与ALS的进展速度呈负相关。事实上，低水平的FoxP3不仅可以预测ALS患者未来的快速进展率，还可以预测存活率下降。在mSOD1小鼠模型中，Tregs的数量在慢性期增加，在此期间小胶质细胞表现为交替激活的(M2)表型。然而，最终失去了神经保护；树突状细胞的数量减少，小胶质细胞转变为经典激活的(M1)表型。初步结果表明，这种转换至少部分是由于有毒的错误折叠的“流氓”蛋白的积累和它们对Toll样受体(Toll-like Receptor，TLR)的激活。在小胶质细胞/运动神经元共培养中加入错误折叠的毒性蛋白SOD1和TDP-43，激活了TLR/CD14信号通路，导致小胶质细胞激活，核因子？b和p38激活，促炎因子(包括NOX2)的释放增加，神经毒性增加。这些有毒的“流氓”蛋白还激活T细胞上的TLRs，抑制其抑制功能。我们假设，阻断Tregs的TLR激活可以显著减缓ALS的疾病。我们的初步结果表明，阻断Tregs的TLR激活显著减缓mSOD1小鼠的疾病。从mSOD1/TLR2-/-小鼠中分离出的CD4+CD25High Tregs在转移到我们的mSOD1/RAG2-/-小鼠中时，显著减缓了疾病的发生，与mSOD1/RAG2-/-小鼠相比，疾病持续时间史无前例地延长了122%或2.24倍。此外，我们已经证明，人类Tregs不仅在mSOD1/RAG2-/-小鼠中存活-因此可以用来评估治疗后的人类Tregs-而且当将其转移到我们的mSOD1/RAG2-/-小鼠中时，在减缓疾病方面与小鼠Tregs一样有效，甚至更有效。我们的目标是使用以下目标来验证我们的新假说：1)通过a)从ALS患者中分离Tregs，将siRNAs导入Tregs，然后评估TLR2和FoxP3的表达，以及b)在体外验证TLR2配体的加入后TLR2的激活降低，并且转基因的Tregs保持其抑制功能来检测Tef的扩展，以确定在降低TLR2活性方面最有效的siRNA。2)通过a)将TLR2抑制的Tregs转移到mSOD1/RAG2-/-小鼠并评估疾病进展，通过a)将TLR2抑制的Tregs转移到我们的mSOD1/RAG2-/-小鼠并评估疾病进展，b)在整个疾病过程中检查这些小鼠的脊髓、淋巴结和血液，以验证这些小鼠在体内是否仍然保持其抑制功能，方法是：a)将TLR2抑制的Tregs转移到mSOD1/RAG2-/-小鼠，b)检查这些小鼠的脊髓、淋巴结和血液是否存在神经损伤、炎症和T细胞。这些实验将导致确定最有效的人类TLR2 siRNAs和治疗方案，可以直接转化为ALS患者。

DESCRIPTION (provided by applicant): Blocking TLR-Activation of Regulatory T cells Slows Disease in ALS Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive neurodegenerative disease. Our recent data demonstrate that regulatory T cells (Tregs) slow disease progression rates in both ALS patients and the mutant (m) SOD1 mouse model of ALS. In ALS patients, numbers of Tregs in blood of rapidly progressing patients are reduced compared to slowly progressing patients; both numbers of Tregs and mRNA expressions of CD25, FoxP3, IL4 and TGF¿ inversely correlate with rate of progression. In fact, low FoxP3 mRNA levels not only predict future rapid progression rates of ALS patients, but also predict reduced survival. In the mSOD1 mouse model, numbers of Tregs are increased during the slow phase, during which time microglia display an alternatively activated (M2) phenotype. However, eventually neuroprotection is lost; numbers of Tregs decline and the microglia switch to a classically activated (M1) phenotype. Preliminary results indicate that this switch may at least partly be due to an accumulation of toxic misfolded "rogue" proteins and their activation of toll-like receptors (TLRs). Misfolded toxic proteins, such as SOD1 and TDP-43, added to microglia/motoneuron co-cultures activated the TLR/CD14 signaling pathway, resulting in microglial activation, NF?B and p38 activation, escalating release of pro-inflammatory factors (including NOX2), and increased neurotoxicity. These toxic "rogue" proteins also activate TLRs on T cells inhibiting their suppressive functions. We hypothesize that blocking TLR activation of Tregs could substantially slow disease in ALS. Our preliminary results demonstrate that blocking TLR-activation of Tregs dramatically slows disease in mSOD1 mice. CD4+CD25High Tregs isolated from mSOD1/TLR2-/- mice dramatically slow disease when transferred into our mSOD1/RAG2-/- mice, extending disease duration an unprecedented 122% or 2.24 fold compared to mSOD1/RAG2-/- mice. In addition, we have demonstrated that human Tregs not only survive in the mSOD1/RAG2-/- mouse - so can be used to evaluate treated human Tregs - but are as effective as, or more than mouse Tregs at slowing disease when transferred into our mSOD1/RAG2-/- mice. Our goal is to test our novel hypothesis using the following aims: 1) To identify the siRNA most effective at reducing TLR2 activation of human Tregs by a) isolating Tregs from ALS patients, transfecting the Tregs with siRNAs and subsequently evaluating TLR2 and FoxP3 expressions, and b) verifying in vitro that TLR2 activation is reduced after addition of TLR2 ligands and that transfected Tregs retain their suppressive functions examining Teff expansion. 2) To verify these siRNA-transfected Tregs retain their suppressive functions in vivo using our mSOD1/RAG2-/- mice by a) transferring the TLR2-inhibited Tregs to our mSOD1/RAG2-/- mice and evaluating disease progression, b) examining the spinal cord, lymph nodes, and blood in these mice throughout disease for neural injury, inflammation, and T cells. These experiments will lead to the identification of the most effective human TLR2 siRNAs and treatment protocols which can be directly translated to ALS patients.