Translational Immunology research: a support for clinical immunological research

转化免疫学研究：临床免疫学研究的支持

• 批准号: 9359924
• 负责人: Massimo Gadina
• 金额: $ 174.34万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 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/9359924
• 关键词: Alleles; Ankylosing spondylitis; Arthritis; Autoantibodies; Autoimmune Diseases; Autoimmune Process; Basic Science; Behcet Syndrome; Biological Assay; Blood Vessels; CRISPR/Cas technology; Cell Line; Cell surface; Cells; Chromosomes, Human, Pair 5; Chronic; Clinical; Clinical Investigator; Clinical Trials; Code; Collaborations; Cooperative Research and Development Agreement; Data; Development; Disease; Endothelium; Epidermis; Erdheim-Chester Disease; Exons; Flow Cytometry; Frameshift Mutation; Generations; Genes; Genetic; Goals; Guide RNA; HLA-B Antigens; HLA-B27 Antigen; Health; Homozygote; Human; IL1A gene; Immune; Immunoassay; Immunologics; Immunology; Immunology procedure; Inbred MRL lpr Mice; Individual; Infiltration; Inflammation; Inflammatory; Instruction; Interleukin-1; Interleukin-6; Journals; Knock-out; Laboratories; Lead; Lipodystrophy; Lipopolysaccharides; Lupus; Manuscripts; Measures; Mediating; Methods; Missense Mutation; Musculoskeletal Diseases; Mutation; National Human Genome Research Institute; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Nature; Nephritis; Nonhomologous DNA End Joining; Nonsense Codon; Organ; Osteoblasts; Patients; Phenotype; Play; Process; Production; Proteins; Publishing; Reporting; Research; Research Personnel; Rheumatism; Role; Serum; Services; Site; Skin; Sorting - Cell Movement; Source; Syndrome; T-Lymphocyte; TNF gene; Techniques; Technology; Temperature; Testing; Translational Research; Ubiquitin; United States National Academy of Sciences; Vasodilation; Western Blotting; autoinflammatory; base; biomarker discovery; clinical application; clinical investigation; cytokine; disorder risk; early onset; expression vector; gain of function mutation; genome-wide; genomic data; induced pluripotent stem cell; inhibitor/antagonist; lymphoblastoid cell line; mRNA Expression; mineralization; molecular marker; monocyte; mouse model; multicatalytic endopeptidase complex; mutant; neutrophil; novel; novel strategies; nuclease; pathogen; pluripotency; protein expression; risk variant; screening; skin barrier; skin disorder; transcriptome sequencing

A major project that involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration with Dan Kastner and Ivona Aksentijevich, we have measured serum cytokines levels in patients with a novel autoinflammatory disease caused by haploinsufficiency of A20 (encoded by TNFAIP3). The syndrome resembles Behcets disease (BD) and our data showed that these patients have increased expression of NFkappaB-mediated proinflammatory cytokines. The manuscript reporting these findings was published in Nature Genetics. In collaboration with Ivona Aksentijevich and Qing Zhou we have reported another novel inflammatory disease caused by the malfunction of otulin, a single gene on chromosome 5. As a result of the mutations, the otulin protein is unable to remove the ubiquitin from various molecules, resulting in an increased production of inflammatory cytokines, ultimately leading to inflammation. The manuscript describing this new syndrome has been published in the Proceedings of the National Academy of Sciences. Furthermore, in collaboration with the Kastner group and Elaine Remmers we have been studying a new Behcets disease-associated loci that we identified using the Immunochip to densely interrogate immune/inflammatory disease loci. We found a noncoding SNP in IL1A associated with disease at genome-wide significance. We found that the disease-associated SNP is also a reported eQTL; the disease risk allele is significantly associated with decreased expression of IL1A in lymphoblastoid cell lines and skin, and the lead disease-associated SNP is also the lead IL1A eQTL SNP. The TIS has been responsible for confirming decreased lipopolysaccharide-stimulated IL1A mRNA expression in healthy control monocytes from disease risk allele homozygotes compared with individuals with 0 or 1 risk allele. Association of the lead disease SNP with IL1B expression was not statistically significant in the published eQTL study. IL-1 is highly expressed in the epidermis and plays an important role in skin barrier functions against pathogens. A manuscript reporting this study is currently being reviewed. With the Goldbach-Mansky group, we studied patients with Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature (CANDLE), a Proteasome-Associated Autoinflammatory Syndrome (PRAAS), who were shown to have mutations in the inducible proteasome-subunit, PSMB8 and PSMB4. The TIS was responsible for measuring patient serum cytokine levels, which revealed an increase in type I IFNs as well as inflammatory cytokines such as IL-6 and TNF. A manuscript was published in the Journal of Clinical Investigation. In collaboration with the Colbert group we have investigated the cytokine profile and the immunologic phenotype of a patient with a de-novo gain-of-function mutation in MyD88, which results in early-onset severe arthritis. We continue to collaborate with NHGRI investigators assessing the cytokine-secreting capabilities of patients with Erdheim-Chester Disease (ECD), a rare, non-familial multisystem disorder characterized by proliferation and infiltration of non-Langerhans histocytes into multiple organs. We compared the cytokine secreted by ECD patients to normal controls after stimulation of PBMCs. The TIS has also been investigating novel approaches for the treatment of autoimmune diseases. In collaboration with the O'Shea group, the Kaplan group, and Pfizer (via a CRADA), we evaluated the efficacy of tofacitinib on a murine model of systemic lupus erythematous (SLE) using the MRL/lpr mouse. Tofacitinib treatment results in a significant amelioration of SLE phenotype, besides reducing autoantibody levels, nephritis, skin inflammation and inflammatory cytokines, tofacitinib mitigates the dysregulation of neutrophil function and induces significant improvements in endothelium-dependent vasorelaxation, which is relevant to vascular health. A manuscript reporting these results was published in Arthritis and Rheumatism. We renewed the CRADA and received second-generation JAK-selective inhibitors, which we are currently evaluating for their activity on T cells. We generated induced pluripotent stem cells (iPSC) from a patient with HLA B-27+ Ankylosing Spondylitis and demonstrated their pluripotency. We then differentiated iPSCs into MSC and osteoblasts, and found that MSC-derived osteoblasts from patients showed greater mineralization capacity compared to normal controls. We have continued the gene-editing project using CRISPR-Cas9 techniques in iPSCs. We have undertaken an effort to generate isogenic human iPS cell lines from AxSpA patients with specific HLA-B*27:04 gene knock-out to assess its role in disease. We used CRISPR/Cas9 gene editing technology to target HLA-B*27:04 through non-homologous end joining by editing the coding sequence at exon 3. We transfected patient-derived iPSCs (HT19) with a CRISPR/Cas9 expression vector containing guide RNA consisting of a 20-nt sequence. The transfected cells were sorted based on GFP expression, and the SURVEYOR nuclease assay was used as a functional test to evaluate Cas9 cutting efficiency. Specifically, we used flow cytometry as a screening method to examine cell surface HLA-B27 expression by developing a method to enrich and screen out knockout or mutant HLA-B27 iPSC colonies by using GFP sorting and single colony selection. RNAseq was then used to identify in-del mutations, frameshifts, missense mutations, or premature stop codons. Two types of mutations were identified by RNAseq on the targeting site of exon 3 of the B27 allele that were predicted to disrupt B27 protein expression. Western Blot results were consistent with the genomic data indicating that B27 protein expression is decreased significantly in the knock-out cell lines. Further studies are currently ongoing to confirm the allele-specific loss of HLA-B expression and to investigate osteoblast development and its function from isogenic B27 knock-out iPS cell lines. The newly generated isogenic HLA-B27 knock-out IPS cell lines are also in the process of being characterized.

涉及TIS的主要项目是自身免疫性和自身炎症性疾病的分子生物标志物的定义。 与Dan Kastner和Ivona Aksentijevich合作，我们测量了由A20的单倍症（由TNFAIP3编码）引起的新型自身炎性疾病患者的血清细胞因子水平。该综合征类似于Behcets病（BD），我们的数据表明，这些患者的表达增加了NFKAPPAB介导的促炎细胞因子。报告这些发现的手稿在自然遗传学中发表。在与伊沃纳·阿克森蒂·杰维奇（Ivona Aksentijevich）和周的合作中，我们报告了另一种新型的炎症性疾病是由5染色体上的单个基因的失功能引起的。由于突变，耳鼻喉蛋白无法从各种分子中去除泛素，导致炎症的产生，最终使炎症症的产生，最终导致炎症，最终导致了炎症的产生。描述这种新综合征的手稿已在美国国家科学院的会议录中发表。 此外，在与Kastner组和Elaine Remmers合作，我们一直在研究使用免疫光学的新型Behcets疾病相关基因座，以密集询问免疫/炎症性疾病基因座。我们发现与疾病有关的IL1A中的非编码SNP具有全基因组意义。我们发现与疾病相关的SNP也是报道的EQTL。疾病风险等位基因与淋巴细胞细胞系和皮肤中IL1A表达的降低显着相关，与铅疾病相关的SNP也是IL1A EQTL SNP的铅。与具有0或1风险等位基因的个体相比，TIS负责确认疾病风险等位基因纯合子的健康对照单核细胞中脂多糖刺激的IL1A mRNA表达降低。在已发表的EQTL研究中，铅疾病SNP与IL1B表达的关联在统计学上没有统计学意义。 IL-1在表皮中高度表达，并且在针对病原体的皮肤屏障功能中起重要作用。目前正在审查该研究的手稿。 在Goldbach-Mansky组的情况下，我们研究了患有慢性非典型嗜中性粒细胞性皮肤病患者，脂肪症和温度升高（蜡烛）是一种蛋白酶体相关的自身炎症综合征（PRAA），这些自身炎症综合征（PRAA）被证明在可诱导的蛋白酶体 - 蛋白酶体 - 纯度，PSMB8和PSMB8和PSMB4和PSMB4。 TIS负责测量患者血清细胞因子水平，这显示I型IFN以及IL-6和TNF等炎性细胞因子的增加。手稿发表在《临床调查杂志》上。 在与Colbert Group合作的情况下，我们研究了MyD88的De-Novo功能获得突变的患者的细胞因子谱和免疫学表型，从而导致早期发作的严重关节炎。 我们继续与NHGRI研究人员合作，评估Erdheim-Chester疾病（ECD）患者的细胞因子分泌能力，这是一种罕见的，非家庭的多系统疾病，其特征在于非兰吉尔人组成细胞扩增和浸润到多个器官中。我们将ECD患者分泌的细胞因子与PBMC刺激后的正常对照组进行了比较。 TIS还一直在研究治疗自身免疫性疾病的新方法。与O'Shea组，Kaplan Group和Pfizer（通过Crada）合作，我们使用MRL/LPR鼠标评估了Tofacitinib在全身性红斑狼疮（SLE）的鼠模型上的功效。 tofacitib的治疗可导致SLE表型的显着改善，除了降低自身抗体水平，肾炎，皮肤炎症和炎症细胞因子外，tofacitinib还可以减轻中性粒细胞功能的失调，并诱导内皮依赖性血管疾病的显着改善，这是相关的。报道这些结果的手稿在关节炎和风湿病中发表。 我们更新了Crada，并接受了第二代JAK选择性抑制剂，目前我们正在评估它们在T细胞上的活性。 我们从HLA B-27+强直性脊柱炎的患者中产生了诱导的多能干细胞（IPSC），并证明了其多能性。然后，我们将IPSC分化为MSC和成骨细胞，发现与正常对照组相比，患者MSC衍生的成骨细胞表现出更大的矿化能力。我们在IPSC中使用CRISPR-CAS9技术继续了基因编辑项目。我们已经努力从特定HLA-B*27：04基因敲除AXSPA患者中产生同源性人IPS细胞系，以评估其在疾病中的作用。我们使用CRISPR/CAS9基因编辑技术来靶向HLA-B*27：04，通过编辑外显子3上的编码序列通过非同源末端连接。我们使用含有CRISPR/CAS9表达矢量的患者衍生的IPSC（HT19），其中包含20-NT序列的指南RNA。根据GFP表达对转染的细胞进行分类，并将测量师核酸酶测定用作评估CAS9切割效率的功能测试。具体而言，我们使用流式细胞仪作为筛选方法来检查细胞表面HLA-B27表达，通过开发一种通过使用GFP分选和单个菌落选择的方法来富集和筛选敲除或突变的HLA-B27 IPSC菌落。然后，将RNASEQ用于识别DEL突变，移架，错义突变或过早的停止密码子。通过RNASEQ在B27等位基因3的外显子3的靶向位点上鉴定出两种类型的突变，这些突变被预测会破坏B27蛋白表达。蛋白质印迹结果与基因组数据一致，表明在敲除细胞系中B27蛋白表达显着降低。目前正在进行进一步的研究，以确认HLA-B表达的等位基因特异性丧失，并研究成骨细胞的发育及其功能。新生成的同源性HLA-B27敲除IPS细胞系也在被表征。