Mechanistic and Therapeutic Role of the TLR4 Signaling Pathway in Type 1 Diabetes

TLR4 信号通路在 1 型糖尿病中的机制和治疗作用

• 批准号: 10718841
• 负责人: ANDREW B HERR
• 金额: $ 70.32万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718841
• 关键词: Acute; Adoptive Transfer; Agonist; Antibodies; Antibody Affinity; Antibody Therapy; Autoimmunity; Avidity; Binding; Biological Assay; Biophysics; Cell Maturation; Cell surface; Cells; Clinical Trials; Cryoglobulins; Data; Diabetes Mellitus; Diabetic Ketoacidosis; Endosomes; Generations; Human; Hyperglycemia; IRF3 gene; IgG1; IgG3; IgG4; Immobilization; Immune; Immunoglobulin Class Switching; Immunologics; Immunology; Inbred NOD Mice; Infiltration; Inflammation; Inflammatory; Insulin; Insulin-Dependent Diabetes Mellitus; Length; Life; Macrophage; Mediating; Molecular; Mus; Myelogenous; Myeloid Cells; Myeloid-derived suppressor cells; Myelopoiesis; Nobel Prize; Paper; Pathway interactions; Patients; Peripheral; Phenotype; Process; Production; Recombinants; Role; Signal Induction; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Structure of beta Cell of islet; Surface; T-Cell Proliferation; T-Lymphocyte; TLR4 gene; Test Result; Testing; Therapeutic; Therapeutic Effect; Tissues; Work; autoimmune pathogenesis; biophysical analysis; biophysical techniques; clinical effect; cytokine; diabetes mellitus therapy; diabetic; glycosylation; humanized mouse; immunoregulation; in vivo; innate immune mechanisms; islet; monocyte; mouse model; novel; prevent; receptor; receptor binding; receptor internalization; therapeutic evaluation; translation to humans

Type 1 Diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta cells. T1D therapies have largely failed in human clinical trials and thus an urgent need exists for targeting novel immune pathways. We have exciting data showing that a TLR4/MD2 agonistic antibody (TLR4-Ab) permanently reversed T1D in 71%, and had a significant clinical effect in 90%, of acutely diabetic non-obese diabetic (NOD) mice. In our recent Diabetes paper, we showed that TLR4-Ab can mobilize and activate myeloid-derived suppressor cells (MDSC) in vivo, that suppress T cells and ameliorate acute T1D upon adoptive transfer. We showed that TLR4- Ab induces TLR4/MD-2 sequestration in endosomes, unlike the canonical TLR4 agonist LPS (which cannot reverse T1D); however, the mechanism by which TLR4-Ab reverses T1D remains unclear. We have now produced anti-human TLR4 antibodies, allowing us to apply these findings to human T1D. Our combined expertise in autoimmunity/T1D (Ridgway), molecular biophysics/Immunology (Herr) and human T1D (Dolan) is well suited to perform these studies. We will pursue three aims: Specific Aim 1. Mechanism of reversal of T1D by TLR4-Ab-induced MDSCs. TLR4-Ab endosomal sequestration may induce prolonged signaling via the TLR4-mediated TRIF pathway, which protects from T1D (2015, Chervonsky et al.). We show that immobilization of TLR4-Ab on a plate, which prevents endosomal sequestration, eliminated TLR4 signaling. Therefore, we hypothesize that TLR4-Ab-induced TLR4/MD2 endosomal sequestration causes sustained TRIF endosomal signaling that induces APCs to undergo MDSC maturation, inducing immune regulation and reversing T1D. Specific Aim 2. Mechanistic role of Fc structure in TLR4-Ab reversal of T1D and cell suppression. While antibody F(ab) structure determines specificity, the Fc region contributes to antibody function through receptor binding and glycosylation. TLR4-Ab is an IgG3 isotype, which has the longest hinge region and increased glycosylation sites. IgG3 antibodies also form cryoglobulins, impacting avidity and internalization. Our preliminary data show that TLR4-Ab F(ab) and F(ab’)2 fragments elicit decreased NFκB signaling compared to full-length TLR4-Ab. This shows a critical role for Fc structure in TLR4-Ab function. Therefore, we hypothesize that the IgG3 Fc portion of the TLR4-Ab is critical to its tolerizing function and we will test this with immunological and molecular biophysical approaches. Specific Aim 3. Testing therapeutic effects of a novel panel of human anti-TLR4 antibodies on human T1D APCs Since the TLR4/MD2 pathway is strongly evolutionarily conserved, we have developed agonistic human recombinant TLR4-Ab (hTLR4-Ab). We show here that these hTLR4-Abs bind to, and activate TLR4/MD2. Our hypothesis is that hTLR4-Ab treatment will induce MDSCs from myeloid precursors and that these huMDSCs will suppress human T-cell proliferation and activation. These studies will characterize novel innate immune mechanisms by which TLR4-Ab treatment can reverse acute T1D, and our studies on the novel human TLR4 antibodies are the first step in translation to human T1D.

1型糖尿病（T1D）是由胰岛素产生胰岛β细胞的自身免疫性破坏引起的。 T1D 在人类临床试验中，疗法在很大程度上失败了，因此迫切需要针对新型免疫 途径。我们有令人兴奋的数据，表明TLR4/MD2激动抗体（TLR4-AB）永久反转 T1D为71％，在90％的急性糖尿病非肥胖糖尿病（NOD）小鼠中具有显着的临床作用。 我们最近的糖尿病纸，我们表明TLR4-AB可以动员和激活髓样衍生的抑制细胞 （MDSC）在体内抑制T细胞并在收养转移时改善急性T1D。我们表明TLR4- 与规范的TLR4激动剂LPS不同，AB在内体中诱导TLR4/MD-2隔离 反向T1D）;但是，TLR4-AB逆转T1D的机制尚不清楚。我们现在有 产生了抗人类TLR4抗体，使我们能够将这些发现应用于人类T1D。我们的结合 自身免疫/T1D（Ridgway），分子生物物理学/免疫学（HERR）和人类T1D（Dolan）的专业知识是 非常适合进行这些研究。我们将追求三个目标：特定目标1。逆转T1D的机制 由TLR4-AB诱导的MDSC。 TLR4-AB内体隔离可以通过 TLR4介导的TRIF途径，可保护T1D（2015，Chervonsky等）。我们表明固定 阻止内体隔离的板上TLR4-AB的ab，消除了TLR4信号传导。因此，我们 假设TLR4-AB诱导的TLR4/MD2内体隔离导致持续的TRIF内体 信号引起APC经历MDSC成熟，诱导免疫调节并逆转T1D。 具体目标2。FC结构在T1D和细胞抑制的TLR4-AB逆转中的机理作用。尽管 抗体F（AB）结构决定了特异性，FC区域通过受体有助于抗体功能 结合和糖基化。 TLR4-AB是IgG3同种型，它具有最长的铰链区域，并且增加 糖基化位点。 IgG3抗体还形成了冷冻球蛋白，从而影响了亲戚和内在化。我们的初步 数据表明，与全长相比 tlr4-ab。这显示了FC结构在TLR4-AB功能中的关键作用。因此，我们假设 TLR4-AB的IgG3 FC部分对于其耐受功能至关重要，我们将通过免疫学和 分子生物物理方法。特定目的3。测试人类小组的治疗作用 由于TLR4/MD2途径的强烈保守，人类T1D APC上的抗TLR4抗体 我们已经开发了激动的人类重组TLR4-AB（HTLR4-AB）。我们在这里表明这些HTLR4-ABS 结合并激活TLR4/MD2。我们的假设是HTLR4-AB治疗将诱导髓样的MDSC 前体和这些HumDSC将抑制人类T细胞的增殖和激活。这些研究会 特征TLR4-AB治疗可以逆转急性T1D的新型先天免疫力学，我们 对新型人类TLR4抗体的研究是转化为人类T1D的第一步。