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（由适用提供）：阻止调节性T细胞的TLR激活减缓ALS肌萎缩性侧面硬化症（ALS）的疾病是一种毁灭性的，快速进行的神经退行性疾病。我们最近的数据表明，ALS患者和ALS的突变体（M）SOD1小鼠模型的调节性T细胞（Treg）疾病进展速率慢。在ALS患者中，与缓慢进展的患者相比，快速进步患者的血液中的Treg数量减少了。 CD25，FOXP3，IL4和TGF¿的Tregs和mRNA表达式的数量与进展速率成反比。实际上，低FOXP3 mRNA水平不仅可以预测ALS患者的未来快速进展率，而且还可以预测存活率降低。在MSOD1小鼠模型中，在缓慢期间的Treg数量增加，在此期间，小胶质细胞显示出替代激活（M2）表型。但是，最终神经保护却丢失了。 Treg的数量下降，小胶质细胞转换为经典激活（M1）表型。初步结果表明，这种转换至少部分是由于有毒的“流氓”蛋白的积累及其激活类似收费的受体（TLR）。添加到小胶质细胞/Motoneuron共培养中的SOD1和TDP-43等有毒蛋白（例如SOD1和TDP-43）激活了TLR/CD14信号通路，从而导致小胶质细胞激活，NF？B和p38激活，逐渐释放了促进感染因素（包括NOX22）和NEUROTOROTOFITY（包括NOUROTOFITY），以及增加的毒性。这些有毒的“流氓”蛋白还激活T细胞上的TLR，抑制其抑制功能。我们假设阻止TLR激活TREG可能会大大减慢ALS疾病的速度。我们的初步结果表明，在MSOD1小鼠中阻止Treg的TLR激活急剧缓慢。与MSOD1/RAG2-/ - 小鼠相比，从MSOD1/TLR2 - / - 小鼠中分离出的CD4+CD25高Tregs从MSOD1/TLR2 - / - 小鼠转移到我们的MSOD1/RAG2 - / - 小鼠中时，疾病急剧缓慢，疾病延长了疾病的持续时间，前所未有的122％或2.24倍。此外，我们已经证明了人treg不仅在MSOD1/rag2 - / - 小鼠中生存，因此可以用来评估经过治疗的人treg，而且在转移到我们的msod1/rag2 - / - 小鼠中时，在缓慢疾病的小鼠treg中也像小鼠Treg一样有效。 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 translated Tregs保留了检查TEFF扩展的抑制功能。 2）使用我们的MSOD1/rag2 - / - 小鼠验证这些siRNA转染的Treg在体内保留其抑制作用，通过a）将TLR2抑制的Treg转移到我们的MSOD1/RAG2/ - / - 小鼠中，并评估脊髓疾病，b）在这些细胞中，淋巴结和流血，这些细胞和流血遍及这些细胞和流血。这些实验将导致鉴定最有效的人类TLR2 siRNA和治疗方案，这些方案可以直接转化为ALS患者。