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

阻断调节性 T 细胞的 TLR 激活可减缓 ALS 疾病

• 批准号: 8491387
• 负责人: Stanley H. Appel
• 金额: $ 19.69万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8491387
• 关键词: Age; Amyotrophic Lateral Sclerosis; Animal Model; Autologous; Blood; CD14 gene; Cells; Clinical Trials; Coculture Techniques; Data; Diagnosis; Disease; Disease Progression; Ensure; Eragrostis; Flow Cytometry; Future; Goals; Human; IL2RA gene; IL4 gene; IL7R gene; In Vitro; Inflammation; Inflammatory; Informed Consent; Injection of therapeutic agent; Lead; Ligands; MAPK14 gene; Manuscripts; Messenger RNA; Methods; Microglia; Motor Neurons; Mus; Neurodegenerative Disorders; Onset of illness; Pathway interactions; Patients; Phase; Phenotype; Play; Proteins; Receptor Activation; Regulatory T-Lymphocyte; Role; Safety; Signal Pathway; Sirolimus; Small Interfering RNA; Spinal Cord; T-Lymphocyte; TLR2 gene; TNF gene; Tail; Testing; Time; Time Study; Toll-like receptors; Transfection; Translating; Treatment Protocols; Veins; Writing; design; effective therapy; illness length; in vivo; lymph nodes; mRNA Expression; mouse model; mutant; nerve injury; neuroprotection; neurotoxicity; novel; protein TDP-43; protein misfolding; public health relevance; research study

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.

描述（由申请人提供）：阻断调节性T细胞的TLR激活减缓ALS中的疾病肌萎缩侧索硬化症（ALS）是一种破坏性的、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（TCLs）减缓ALS患者和ALS突变型（m）SOD 1小鼠模型的疾病进展速度。在ALS患者中，与缓慢进展的患者相比，快速进展的患者血液中的TGFm的数量减少; TGFm的数量和CD 25、FoxP 3、IL 4和TGF β的mRNA表达与进展速率呈负相关。事实上，低FoxP 3 mRNA水平不仅预测ALS患者未来的快速进展率，而且预测生存率降低。在mSOD 1小鼠模型中，在缓慢期期间，TcM的数量增加，在此期间，小胶质细胞显示出交替激活（M2）表型。然而，最终神经保护作用丧失; T细胞数量下降，小胶质细胞转变为经典激活（M1）表型。初步结果表明，这种转换可能至少部分是由于有毒的错误折叠的“流氓”蛋白质的积累和它们对Toll样受体（TLR）的激活。错误折叠的毒性蛋白，如SOD 1和TDP-43，加入到小胶质细胞/运动神经元共培养激活TLR/CD 14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NOX 2）释放升级，神经毒性增加。这些有毒的“流氓”蛋白也激活T细胞上的TLR，抑制其抑制功能。我们假设阻断TLR激活TGFAP可以显著减缓ALS的疾病。我们的初步研究结果表明，阻断TLR激活TcR可显著减缓mSOD 1小鼠的疾病。从mSOD 1/TLR 2-/-小鼠中分离的CD 4 + CD 25 High T细胞在转移到我们的mSOD 1/RAG 2-/-小鼠中时显著减缓了疾病，与mSOD 1/RAG 2-/-小鼠相比，疾病持续时间延长了前所未有的122%或2.24倍。此外，我们已经证明，人TCL 4不仅在mSOD 1/RAG 2-/-小鼠中存活-因此可以用于评估治疗的人TCL 4-，而且当转移到我们的mSOD 1/RAG 2-/-小鼠中时，在减缓疾病方面与小鼠TCL 4一样有效或更有效。我们的目标是使用以下目标来测试我们的新假设：a）从ALS患者中分离TLR 2，用siRNA转染TLR 2，随后评估TLR 2和FoxP 3表达，和B）在体外证实加入TLR 2配体后TLR 2活化减少，并且转染的TCLs保留其抑制功能，Teff膨胀2)为了使用我们的mSOD 1/RAG 2-/-小鼠验证这些siRNA转染的TCL 4在体内保留其抑制功能，通过a）将TLR 2抑制的TCL 4转移到我们的mSOD 1/RAG 2-/-小鼠并评估疾病进展，B）在这些小鼠的整个疾病过程中检查脊髓、淋巴结和血液中的神经损伤、炎症和T细胞。这些实验将导致最有效的人类TLR 2 siRNA的鉴定和治疗方案，可以直接翻译给ALS患者。