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: 8635400
• 负责人: Stanley H. Appel
• 金额: $ 23.39万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8635400
• 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; Interleukin-1; 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 激活可减缓 ALS 疾病肌萎缩侧索硬化症 (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 (Treg) 可以减缓 ALS 疾病的进展速度。 ALS 患者和 ALS 突变 (m) SOD1 小鼠模型中，与缓慢进展的患者相比，快速进展的患者血液中的 Tregs 数量均减少； Tregs 数量以及 CD25、FoxP3、IL4 和 TGF 的 mRNA 表达事实上，低 FoxP3 mRNA 水平不仅可以预测 ALS 患者未来的快速进展率，还可以预测在 mSOD1 小鼠模型中，Treg 的数量在缓慢阶段增加。小胶质细胞表现出替代激活（M2）表型，但最终神经保护作用会丧失；Treg 数量下降，小胶质细胞会转变为经典激活（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“流氓”蛋白的积累及其对 Toll 样受体 (TLR) 的激活将错误折叠的有毒蛋白（例如 SOD1 和 TDP-43）添加到小胶质细胞/运动神经元共培养物中会激活 TLR/CD14 信号通路，导致小胶质细胞激活、NF?B 和 p38 激活、促炎因子（包括 NOX2）释放增加以及神经毒性增加。这些有毒的“流氓”蛋白也会激活。 T 细胞上的 TLR 抑制其抑制功能。我们的初步结果表明，阻断 TLR 激活可显着减缓从 mSOD1/mSOD1 小鼠中分离的 TLR 疾病。当 TLR2-/- 小鼠转移到我们的 mSOD1/RAG2-/- 小鼠体内时，疾病持续时间显着减缓，与传统小鼠相比，疾病持续时间史无前例地延长了 122% 或 2.24 倍。此外，我们还证明人类 Tregs 不仅能在 mSOD1/RAG2-/- 小鼠体内存活，因此可用于评估治疗后的人类 Tregs，而且与小鼠一样有效，甚至更有效。当转移到我们的 mSOD1/RAG2-/- 小鼠中时，Treg 可以减缓疾病我们的目标是使用以下目标来测试我们的新假设：1) 确定最有效地减少人类 TLR2 激活的 siRNA。通过 a) 从 ALS 患者中分离 Tregs，用 siRNA 转染 Tregs，然后评估 TLR2 和 FoxP3 表达，b) 体外验证添加 TLR2 配体后 TLR2 激活减少，并且检查 Teff 扩增，转染的 Tregs 保留其抑制功能2) 使用我们的 mSOD1/RAG2-/- 小鼠通过 a) 转移来验证这些 siRNA 转染的 Tregs 在体内保留其抑制功能。 b) 在整个疾病过程中检查这些小鼠的脊髓、淋巴结和血液中的神经损伤、炎症和 T 细胞。确定最有效的人类 TLR2 siRNA 和可直接应用于 ALS 患者的治疗方案。

项目成果

Immune-mediated mechanisms in the pathoprogression of amyotrophic lateral sclerosis.

• DOI: 10.1007/s11481-013-9489-x
• 发表时间: 2013-09
• 期刊: JOURNAL OF NEUROIMMUNE PHARMACOLOGY
• 影响因子: 6.2
• 作者: Zhao, Weihua;Beers, David R.;Appel, Stanley H.
• 通讯作者: Appel, Stanley H.