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

肌萎缩性侧索硬化症（ALS）是一种毁灭性的、快速进展的神经退行性疾病。我们最近的数据表明，调节性T细胞（Tregs）减缓了ALS患者和突变(m) SOD1小鼠ALS模型的疾病进展速度。在ALS患者中，与进展缓慢的患者相比，进展迅速的患者血液中treg的数量减少；Tregs的数量以及CD25、FoxP3、IL4和TGF - mRNA的表达与进展速度呈负相关。事实上，低FoxP3 mRNA水平不仅预示着ALS患者未来的快速进展率，也预示着生存期的降低。在mSOD1小鼠模型中，treg的数量在慢期增加，在此期间小胶质细胞表现出交替激活（M2）表型。然而，神经保护最终会丧失；treg数量下降，小胶质细胞切换到经典激活（M1）表型。初步结果表明，这种转变可能至少部分是由于有毒错误折叠的“流氓”蛋白质的积累及其toll样受体（TLRs）的激活。错误折叠的毒性蛋白，如SOD1和TDP-43，加入到小胶质细胞/运动神经元共培养中，激活TLR/CD14信号通路，导致小胶质细胞活化，NF？B和p38激活，促炎因子（包括NOX2）释放增加，神经毒性增加。这些有毒的“流氓”蛋白质也会激活T细胞上的tlr，抑制它们的抑制功能。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tlr -激活Tregs可显著减缓mSOD1小鼠的疾病。从mSOD1/TLR2-/-小鼠中分离的CD4+CD25High Tregs转移到我们的mSOD1/RAG2-/-小鼠后，显著减缓了疾病，与mSOD1/RAG2-/-小鼠相比，疾病持续时间延长了122%或2.24倍。此外，我们已经证明，人类treg不仅在mSOD1/RAG2-/-小鼠中存活——因此可以用于评估治疗过的人类treg——而且当转移到mSOD1/RAG2-/-小鼠中时，在减缓疾病方面与小鼠treg一样有效，甚至比小鼠treg更有效。我们的目标是通过以下目的来验证我们的新假设：1)通过a)从ALS患者中分离treg，用siRNA转染treg并随后评估TLR2和FoxP3的表达，确定最有效地降低人类treg TLR2激活的siRNA； b)在体外验证添加TLR2配体后TLR2激活降低，并且通过检查Teff扩增，转染的treg保留其抑制功能。2)利用mSOD1/RAG2-/-小鼠，通过a)将tlr2抑制的Tregs转移到mSOD1/RAG2-/-小鼠并评估疾病进展，b)检查这些小鼠在疾病期间的脊髓、淋巴结和血液中的神经损伤、炎症和T细胞，验证这些sirna转染的Tregs在体内保留其抑制功能。这些实验将导致鉴定最有效的人类TLR2 sirna和治疗方案，可以直接翻译为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.