Effect Of Cytokines In Host Defense And Inflammation

细胞因子在宿主防御和炎症中的作用

• 批准号: 7964281
• 负责人: JOHN I GALLIN
• 金额: $ 24.61万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 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/7964281
• 关键词: Antibodies; B-Lymphocytes; Binding; Biochemical; Biological Assay; Biological Response Modifiers; Bone Marrow; Bulla; CD34 gene; Cells; Childhood; Chronic; Chronic Granulomatous Disease; Contractor; Development; Devices; Disease; Drug Design; Endotoxins; Ensure; Enzymes; Evolution; Experimental Models; Exudate; Fibroblasts; Forearm; Formalin; Functional RNA; Future; Gene Expression; Generations; Genome; Goals; Granuloma; Host Defense; Human; Human Volunteers; IL8 gene; Immune; Immunofluorescence Immunologic; Immunologic Deficiency Syndromes; In Vitro; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Interleukin-1; Interleukin-10; Job' s Syndrome; Laboratories; Link; Literature; Liver; MAP Kinase Gene; MAPK14 gene; Mediating; Mediator of activation protein; Membrane; MicroRNAs; Microarray Analysis; Molecular; Mutation; Myeloid Progenitor Cells; NADPH Oxidase; Nuclear; Organism; Paraffin Embedding; Pathway interactions; Patients; Phagocytes; Phosphorylation; Play; Preparation; Production; Protocols documentation; RNA; RNA Sequences; Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Sarcoidosis; Signal Transduction; Signal Transduction Pathway; Skin; Spleen; Stem Cell Development; Structure of parenchyma of lung; Superoxides; Surveys; System; TLR4 gene; Techniques; Technology; Tissues; Toll-Like Receptor Pathway; Translations; Variant; Work; antimicrobial peptide; base; chronic graft versus host disease; cytokine; cytotoxic; human IRAK4 protein; human subject; in vivo; inhibitor/antagonist; monocyte; neutrophil; neutrophil cytosol factor 67K; novel; p21 activated kinase; peripheral blood; research study; response

In 2009 we continued our accrual of patients into our blister study protocol. The protocol involves the formation of blisters on the forearms of human volunteers to study the inflammatory response in vivo. This year, we intiated a project comparing the transcriptomes of peripheral blood neutrophils with matched inflammatory exudate (blister) neutrophils from seven normal donors. For this project, we have teamed with the Rocky Mountain Laboraty Research Technologies Branch (RML) who will provide expert microarray analysis of these samples. We are increasing the number of chronic GvHD patients in our study pool (now a total of 4 patients) as they become available from our collaborators in Dr. Harry Malech's laboratory. Also, with the help of LCID, we have now studied a total of five patients with Job's syndrome and are awaiting quantitative analysis by our contractor at SAIC-Frederick. (Zarember 20% effort). In addition to several normals, we have also studied three patients with chronic graft-versus-host disease and two patients with Jobs Syndrome. Additional patients will be required to fully power these studies to determine whether the observed dysregulation of cytokine production is statistically significant (Kol Zarember, 20% effort). In 2009 we continued to study the molecular basis for priming and activation of the NADPH oxidase and superoxide production by endotoxin (LPS). The NADPH oxidase (NOX), an oligomeric enzyme, plays a key role in polymorphonuclear neutrophil (PMN)-mediated host defense by producing cytotoxic superoxide anion (O2.-). Whereas in vitro and biochemical studies have examined the assembly and activation of this important host immune defense system, few studies have examined the function of NOX in human patients with primary immunodeficiencies other than chronic granulomatous disease. We studied the activation of NOX in PMN from patients with two distinct immunodeficiencies, interleukin-1 receptor associated kinase 4 (IRAK4) deficiency and nuclear factor kappa (NF-κB) essential modulator (NEMO or IKKγ) deficiency. We observed impaired O2.- generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4. NEMO-deficient PMN also generated less O2.- in response to LPS and fMLP and translocated less p47phox and p67phox to membranes than normal PMN but were more responsive than IRAK4-deficient cells. Decreased LPS and fMLP induced phosphorylation of p38 MAPK in both IRAK4- or NEMO-deficient PMN and of p21-activated kinases (PAK) in IRAK4-deficiency implicates additional signal transduction pathways in regulating PMN superoxide activation by LPS and fMLP (Anjali Singh, 85% effort; Kol Zarember 10% effort). In 2009 we initiatied a study of granuloma formation utilizing immunofluorescence to probe the various cellular and molecular components of granulomas. Using this technique, we will demonstrate the various types of immune cells (including neutrophils, monocytes, T and B cells, and fibroblasts) and immune mediators (such as IL-1, IL-8, IL-10, TGF, and various antimicrobial peptides) present in granulomas and potentially contributing to their formation. We have validated the antibodies against many of these targets in peripheral blood smears, a formalin fixed, paraffin embedded buffy coat pellet, and normal formalin fixed, paraffin embedded spleen and bone marrow sections to ensure that the desired binding is achieved with minimal background/non-specific binding. Experiments are underway using liver, spleen, and lung tissue from patients with CGD that contain granulomas and efforts are being made to obtain granuloma-containing tissue from patients with other diseases such as inflammatory bowel disease and sarcoidosis. (Soule, 30% effort). This past year we initiated another project to better understand the potential of microRNA in the regulation of phagocyte function, including regulation of production of cytokines. MicroRNAs comprise a class of over 500 small, non-coding RNA that regulate gene expression by inhibiting RNA translation. They are felt to regulate the expression of over 30% of the genome and specific microRNA species have been shown to be active in neutrophils. A complete survey of the microRNA species in neutrophils and monocytes has not been performed. We have used several commercially available RT-PCR kits to assay specific microRNA in neutrophils, however this has been complicated by significant experiment to experiment variation. In response to this, we have been collaborating with the Rocky Mountain Laboratory to perform whole RNA sequencing on total RNA isolated from purified neutrophils preparations from normal controls and patients with both X-linked (gp91-deficient) and autosomal recessive (p47-deficient) CGD patients. This information will itself be novel, but will also facilitate the development of future studies involving both microRNA microarrays and conventional RT-PCR. We have also begun preliminary work studying the viability of transfecting microRNA precursors and inhibitors into neutrophils to determine the effect of altering microRNA expression on neutrophil function. Finally, we have been speaking with other investigators about the possibility of studying the role of microRNA in myeloid stem cells (CD34+ cells). There is a large body of literature demonstrating the role of microRNA in development at the cellular and organism level. It is likely that microRNA are active in directing myeloid stem cell development and differentiation, but this has not been extensively studied.

2009 年，我们继续招募患者加入我们的泡罩研究方案。该方案涉及在人类志愿者的前臂上形成水泡，以研究体内炎症反应。 今年，我们启动了一个项目，比较外周血中性粒细胞的转录组与来自七个正常捐赠者的匹配的炎症渗出物（水泡）中性粒细胞。 对于这个项目，我们与落基山实验室研究技术分部 (RML) 合作，后者将为这些样本提供专家微阵列分析。 我们正在增加研究池中慢性 GvHD 患者的数量（现在共有 4 名患者），因为我们的合作者可以从 Harry Malech 博士的实验室获得这些患者。 此外，在 LCID 的帮助下，我们现在已经研究了总共 5 名乔布斯综合症患者，正在等待 SAIC-Frederick 承包商的定量分析。 （扎伦伯 20% 的努力）。除了几名正常人之外，我们还研究了三名慢性移植物抗宿主病患者和两名乔布斯综合症患者。 将需要更多患者来充分支持这些研究，以确定观察到的细胞因子产生失调是否具有统计学意义（Kol Zarember，20％的努力）。 2009 年，我们继续研究 NADPH 氧化酶启动和激活以及内毒素 (LPS) 产生超氧化物的分子基础。 NADPH 氧化酶 (NOX) 是一种寡聚酶，通过产生细胞毒性超氧阴离子 (O2.-)，在多形核中性粒细胞 (PMN) 介导的宿主防御中发挥关键作用。虽然体外和生化研究已经检验了这一重要宿主免疫防御系统的组装和激活，但很少有研究检验 NOX 在除慢性肉芽肿性疾病以外的原发性免疫缺陷人类患者中的功能。我们研究了患有两种不同免疫缺陷的患者中 PMN 中 NOX 的激活，即白细胞介素 1 受体相关激酶 4 (IRAK4) 缺陷和核因子 kappa (NF-κB) 必需调节剂（NEMO 或 IKKγ）缺陷。我们观察到 LPS 处理和 fMLP 激活的 IRAK4 缺陷型 PMN 产生 O2.- 受损，这与 p47phox 磷酸化降低以及 p47phox、p67phox、Rac2 和 gp91phox/Nox2 向膜的异常易位相关，表明 NOX 激活途径的 TLR4 信号传导需要 IRAK4。与正常 PMN 相比，NEMO 缺陷型 PMN 在响应 LPS 和 fMLP 时产生的 O2.- 也较少，并且将较少的 p47phox 和 p67phox 转移到膜上，但比 IRAK4 缺陷型细胞的反应性更强。 LPS 和 fMLP 的减少诱导了 IRAK4 或 NEMO 缺陷型 PMN 中 p38 MAPK 的磷酸化，以及 IRAK4 缺陷型中 p21 激活激酶 (PAK) 的磷酸化，这表明 LPS 和 fMLP 调节 PMN 超氧化物激活过程中存在额外的信号转导途径（Anjali Singh，85% 努力；Kol Zarember 10% 努力）。 2009 年，我们启动了一项肉芽肿形成研究，利用免疫荧光来探测肉芽肿的各种细胞和分子成分。 利用这项技术，我们将展示肉芽肿中存在的各种类型的免疫细胞（包括中性粒细胞、单核细胞、T 细胞和 B 细胞以及成纤维细胞）和免疫介质（例如 IL-1、IL-8、IL-10、TGF 和各种抗菌肽），并可能有助于肉芽肿的形成。 我们已经在外周血涂片、福尔马林固定、石蜡包埋的血沉棕黄层颗粒和正常福尔马林固定、石蜡包埋的脾脏和骨髓切片中验证了针对许多这些靶标的抗体，以确保以最小的背景/非特异性结合实现所需的结合。 正在进行的实验使用来自 CGD 患者的含有肉芽肿的肝脏、脾脏和肺组织，并且正在努力从患有其他疾病（例如炎症性肠病和结节病）的患者中获取含有肉芽肿的组织。 （灵魂，30%的努力）。 去年，我们启动了另一个项目，以更好地了解 microRNA 在调节吞噬细胞功能（包括调节细胞因子的产生）方面的潜力。 MicroRNA 包含一类 500 多种小非编码 RNA，它们通过抑制 RNA 翻译来调节基因表达。 人们认为它们调节超过 30% 的基因组的表达，并且特定的 microRNA 种类已被证明在中性粒细胞中具有活性。 尚未对中性粒细胞和单核细胞中的 microRNA 种类进行完整的调查。 我们已经使用了几种市售的 RT-PCR 试剂盒来测定中性粒细胞中的特定 microRNA，然而，由于实验之间的重大差异，这变得复杂化。 为此，我们一直与落基山实验室合作，对从正常对照和 X 连锁（gp91 缺陷）和常染色体隐性遗传（p47 缺陷）CGD 患者的纯化中性粒细胞制剂中分离出的总 RNA 进行全 RNA 测序。 这些信息本身是新颖的，但也将促进未来涉及 microRNA 微阵列和传统 RT-PCR 的研究的发展。 我们还开始了初步研究将 microRNA 前体和抑制剂转染至中性粒细胞的可行性，以确定改变 microRNA 表达对中性粒细胞功能的影响。 最后，我们一直在与其他研究人员讨论研究 microRNA 在骨髓干细胞（CD34+ 细胞）中的作用的可能性。 有大量文献证明了 microRNA 在细胞和生物体水平发育中的作用。 microRNA 很可能在指导骨髓干细胞发育和分化方面发挥着积极作用，但这尚未得到广泛研究。