Pathogenic Mechanisms in Spondyloarthritis

脊柱关节炎的发病机制

基本信息

批准号：
9563902
负责人：
Robert Colbert
金额：
$ 358.22万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9563902
关键词：
ANTXR2 gene Adaptor Signaling Protein Address Adipocytes Adolescent Affect Alleles Animal Model Ankylosing spondylitis Arthritis Autophagocytosis Back Biopsy Cell Differentiation process Cell Lineage Cell surface Cells Cellular Stress Child Childhood Chronic Childhood Arthritis Classification Clinical Colitis Complex Development Dimerization Disease Disulfides Endoplasmic Reticulum Environmental Risk Factor Exhibits Fibroblasts Future Gene Expression Gene Expression Profiling Genes Genetic Genetic study Goals HLA-B Antigens HLA-B27 Antigen HLA-B7 Antigen Hematopoietic Heritability Human IL12B gene Immunologic Deficiency Syndromes Impairment Incidence Individual Inflammation Interleukin-1 Interleukin-1 Receptors Interleukin-17 Knock-out Knowledge Laboratories Link Location Lymphoma Mesenchymal Stem Cells Minerals Modeling Mutation Myeloid Cells Orchitis Osteoblasts Osteogenesis Outcome Pathogenesis Pathogenicity Pathway interactions Patients Peptides Pharmacology Phenotype Play Population Predisposition Prevalence Publications Quality Control Rat Strains Rattus Receptor Signaling Reporting Research Rheumatology Risk Role STAT3 gene Severities Single-Gene Defect Sirolimus Skin Spondylarthritis Symbiosis Symptoms TYK2 Toll-like receptors Ubiquitination Variant Virus Work bone cell type chemokine cytokine design dimer early onset endoplasmic reticulum stress experimental study functional genomics gain of function gain of function mutation gastrointestinal genetic variant genome editing genome-wide gut microbiome gut microbiota imaging genetics improved induced pluripotent stem cell interest interleukin-23 loss of function loss of function mutation mTOR Inhibitor macrophage meetings microbial microbial community microbiome microbiota mutant neutrophil non-genetic overexpression prevent progenitor rare variant risk variant tool transcriptome sequencing

项目摘要

Induced Pluripotent Stem Cells Reveal Ankylosing Spondylitis Risk Gene Expression in Bone Progenitors Aberrant bone formation in AS is poorly understood, and involves cell types that are not readily accessible for study. To circumvent this obstacle one goal of our research has been to develop patient-derived induced pluripotent stem cells (iPSCs) that can be differentiated into mesenchymal stem cells (MSCs) and their derivatives including bone-forming osteoblasts. We demonstrated the feasibility of reprogramming skin fibroblasts obtained from shave skin biopsies into iPSCs using non-integrating virus-encoded factors, and have differentiated these cells into multiple disease-relevant cell lineages including MSCs, mineralizing osteoblasts, adipocytes, and myeloid cells. Genome-wide gene expression analysis using RNA-Seq shows that the expression of several AS risk genes is enriched in MSCs, with some also prominent in iPSCs, supporting the utility of this approach. Preliminary studies suggest that osteoblasts derived from patients with AS mineralize to a greater extent than cells from healthy controls. Experiments designed to understand the mechanism underlying this effect are underway. The ability to generate iPSCs provides a powerful tool to explore the functional genomics of AS risk genes that may impact bone formation and other aspects of disease pathogenesis. The Interaction Between HLA-B27 and ERAP1 in Spondyloarthritis Genetic studies point toward loss-of-function of ERAP1 as a protective factor for the development of AS in HLA-B27 positive individuals. The mechanism underlying this effect is not known. To further assess HLA-B27-ERAP1 interactions in disease, we produced a functional ERAP1 knockout in rats using genome editing. Preliminary results show that ERAP1 deficiency confers partial protection from the development of experimental spondyloarthritis caused by HLA-B27 expression in rats. ERAP1 deficiency reduced the prevalence of arthritis and orchitis by two thirds, while gastrointestinal inflammation was not reduced. ERAP1 deficiency increases presentation of longer peptides and also promotes the formation of disulfide-linked dimers of HLA-B27 on the cell surface. In contrast, ERAP1 deficiency reduces the accumulation of misfolded disulfide-linked dimers and oligomers in the endoplasmic reticulum (ER), and mitigates the development of ER stress. Overall, ERAP1 deficiency improves HLA-B27 folding and reduces misfolding, while it impairs the folding of another allele (HLA-B7). Current studies are focused on determining whether reduced ER stress and its consequences explain effects of ERAP1 deficiency on the spondyloarthritis phenotype, and whether this pathway can be targeted to reduce the incidence and severity of experimental spondyloarthritis in rats. The Role of Autophagy in the Degradation of Misfolded HLA-B27 Heavy Chains The propensity of HLA-B27 to misfold can generate cellular stress when sufficient heavy chains accumulate in the ER. Cells can eliminate misfolded heavy chains through a quality control pathway known as ER-associated degradation (ERAD), but this appears to be insufficient to prevent HLA-B27 from accumulating and generating ER stress in rat macrophages. We found that autophagy is also involved in the degradation of HLA-B27 heavy chains, although it is also insufficient to prevent ER stress. However, further augmentation of this pathway with the pharmacologic inhibitor of mTOR rapamycin promptly increases the degradation of misfolded HLA-B27. Moreover, ubiquitination of HLA-B27 heavy chains in the ER was inefficient compared to another allele (HLA-B7) that does not misfold, identifying this as a potential cause of inefficient ERAD. This work has been submitted for publication and is under revision. Genetic or pharmacologic manipulation of the autophagy pathway may be exploited to alleviate the consequences of HLA-B27 misfolding. The Microbiome and Spondyloarthritis Gut microbiota are thought to play a critical role in the development of spondyloarthritis. We reported previously that HLA-B27 expression alters the gut microbiome in a rat model of human spondyloarthritis. We asked whether specific microbial communities altered by HLA-B27 were linked with the development of colitis in rats with experimental spondyloarthritis. Surprisingly, we found that gut microbial communities altered by HLA-B27 were very different in 3 strains of rats housed in two different locations. Thus, genetic and environmental factors play a large role in determining effects of HLA-B27. These preliminary results have been presented at rheumatology and microbiome meetings and submitted for publication. We are currently investigating common functions of the diverse microbiota, and plan to build on these studies to better understand how commensal microbiota affect spondyloarthritis pathogenesis. Genetic Contributions to Early Onset Severe Juvenile Arthritis MyD88 is a critical adaptor protein for TLR and IL-1 receptor signaling. Loss-of-function mutations in MyD88 cause severe immunodeficiency, while somatic gain-of-function mutations have been linked to certain lymphomas. We discovered a de novo germline MYD88 mutation in a child with destructive polyarticular juvenile arthritis, and have found that the mutation increases MyD88 oligomerization and NF-B activation. Cells expressing mutant MyD88 overexpress chemokines and cytokines, and some hyper-respond to IL-1. Culture supernatants exhibit strikingly enhanced neutrophil chemotactic activity. Thus, this germline MyD88 mutation produces gain-of-function effects in hematopoietic as well as non-hematopoietic cells that are likely to contribute to the development of arthritis. This work has been presented in abstract form at national meetings, and is under review for publication. Future studies will focus on how the mutation leads to a complex arthritis phenotype. These results support a role for single gene defects contributing to the pathogenesis of JIA.

诱导的多能干细胞揭示了骨祖细胞中的连接性脊柱炎风险基因表达在AS中的异常骨形成的理解很少，并且涉及不容易访问研究的细胞类型。为了避免这一障碍，我们研究的一个目标是开发患者衍生的诱导多能干细胞（IPSC），这些干细胞（IPSC）可以分化为间充质干细胞（MSC）及其衍生物，包括骨形成骨成骨细胞。我们证明了使用非整合病毒编码的因子将从剃须皮活检获得的皮肤成纤维细胞重新编程的皮肤成纤维细胞的可行性，并将这些细胞区分为多种疾病相关的细胞谱系，包括MSC，包括MSSC，矿化成骨细胞，脂肪细胞和骨髓细胞。使用RNA-Seq的全基因组基因表达分析表明，几种作为风险基因的表达富含MSC，其中一些在IPSC中也很突出，从而支持这种方法的实用性。初步研究表明，与健康对照细胞相比，矿物质矿物质的患者衍生出的成骨细胞。旨在了解这种效果的机制的实验正在进行中。产生IPSC的能力为探索AS的功能基因组学提供了强大的工具，该功能基因组可能影响骨骼形成和疾病发病机理的其他方面。 HLA-B27与ERAP1之间的相互作用在脊椎关节炎中遗传研究指出，ERAP1的功能丧失是HLA-B27阳性个体中AS发展的保护因素。这种效果的基础机制尚不清楚。为了进一步评估疾病中的HLA-B27-ERAP1相互作用，我们使用基因组编辑在大鼠中产生了功能性ERAP1敲除。初步结果表明，ERAP1缺乏症赋予部分保护，免受由大鼠HLA-B27表达引起的实验性脊椎关节炎的发展。 ERAP1缺乏症将关节炎和提供佛炎的患病率降低了三分之二，而胃肠道炎症并未减少。 ERAP1缺乏增加了较长肽的表现，还促进了细胞表面上HLA-B27的二硫键连接二聚体的形成。相反，ERAP1缺乏症减少了内质网（ER）中错误折叠的二硫键二聚体和低聚物的积累，并减轻了ER应力的发展。总体而言，ERAP1缺乏可改善HLA-B27折叠并减少错误折叠，而它会损害另一个等位基因的折叠（HLA-B7）。当前的研究集中在确定降低的ER应力及其后果是否解释了ERAP1缺乏对脊柱蛋白酶炎表型的影响，以及该途径是否可以针对降低大鼠实验性脊柱炎的发生率和严重程度。自噬在错误折叠的HLA-B27重链降解中的作用当足够的重链积累在ER中时，HLA-B27对错误折叠的倾向会产生细胞应激。细胞可以通过称为ER相关降解（ERAD）的质量控制途径消除错误折叠的重链，但这似乎不足以防止HLA-B27在大鼠巨噬细胞中积累和产生ER应力。我们发现自噬也参与了HLA-B27重链的降解，尽管它也不足以防止ER应力。但是，使用MTOR雷帕霉素的药理学抑制剂进一步扩大了这一途径，迅速增加了错误折叠的HLA-B27的降解。此外，与另一个没有错误折叠的等位基因（HLA-B7）相比，ER中HLA-B27重链的泛素化效率低下，认为这是效率低下的潜在原因。这项工作已提交出版，并正在修订中。可以利用自噬途径的遗传或药理操作，以减轻HLA-B27错误折叠的后果。微生物组和脊椎关节炎肠道微生物群被认为在脊椎关节炎的发展中起着至关重要的作用。我们先前报道说，HLA-B27表达会改变人脊椎关节炎大鼠模型中的肠道微生物组。我们询问HLA-B27改变的特定微生物群落是否与实验性脊椎关节炎的大鼠结肠炎的发展有关。令人惊讶的是，我们发现在两个不同位置容纳的大鼠中，由HLA-B27改变的肠道微生物群落截然不同。因此，遗传和环境因素在确定HLA-B27的影响方面起着重要作用。这些初步结果已在风湿病学和微生物组会议上提出，并提交出版。我们目前正在研究各种微生物群的共同功能，并计划基于这些研究，以更好地了解共生微生物群如何影响脊椎关节炎的发病机理。对早期发作的遗传贡献 MyD88是用于TLR和IL-1受体信号传导的关键衔接蛋白。 MyD88的功能丧失突变会导致严重的免疫缺陷，而运动获得突变已与某些淋巴瘤有关。我们在患有破坏性多关节少年关节炎的儿童中发现了从头种系Myd88突变，发现该突变会增加MyD88的低聚和NF-B激活。表达突变MyD88过表达的趋化因子和细胞因子的细胞，有些对IL-1的反应。培养上清液表现出明显增强的中性粒细胞趋化活性。因此，该种系MyD88突变会在造血和非造血细胞中产生功能的效果，这些细胞可能有助于关节炎的发展。这项工作是在国家会议上以抽象形式提出的，并正在审查出版。未来的研究将集中于突变如何导致复杂的关节炎表型。这些结果支持导致JIA发病机理的单个基因缺陷的作用。